Although in his later years Muddy usually said that he was born in Rolling Fork, Mississippi in 1915, he was actually born at Jug's Corner in neighboring Issaquena County, Mississippi. Recent research has uncovered documentation showing that in the 1930s and 1940s he had reported his birth year as 1913 on both his marriage license and musicians' union card. A 1955 interview in the Chicago Defender is the earliest claim of 1915 as his year of birth, which he continued to use in interviews from that point onward. On the other hand, the 1920 census lists him as five years old as of March 6, 1920, suggesting that his birth year may have been 1914. The Social Security Death Index, relying on his Social Security card application, lists him as being born April 5, 1915.

His grandmother Della Grant raised him after his mother died shortly after his birth. His fondness for playing in mud earned him the nickname "Muddy" at an early age. He later changed it to "Muddy Water" and finally "Muddy Waters". He started out on harmonica but by age seventeen he was playing the guitar at parties emulating two blues artists who were extremely popular in the south, Son House and Robert Johnson. "His thick heavy voice, the dark coloration of his tone and his firm, almost solid, personality were all clearly derived from House," wrote music critic Peter Guralnick in Feel Like Going Home, "but the embellishments which he added, the imaginative slide technique and more agile rhythms, were closer to Johnson."

On November 20, 1932 Muddy married Mabel Berry; Robert Nighthawk played guitar at the wedding, and the party reportedly got so wild the floor fell in. Mabel left Muddy three years later when Muddy's first child was born - the child's mother was Leola Spain, sixteen years old, "married to a man named Steve" and "going with a guy named Tucker". Leola was the only one of his girlfriends with whom Muddy would stay in touch throughout his life; they never married. By the time he finally cut out for Chicago in 1943, there was another Mrs. Morganfield left behind, a girl called Sallie Ann.

In 1940, Muddy moved to Chicago for the first time. He played with Silas Green a year later, and then returned to Mississippi. In the early part of the decade he ran a juke joint, complete with gambling, moonshine and a jukebox; he also performed music there himself. In the summer of 1941 Alan Lomax went to Stovall, Mississippi, on behalf of the Library of Congress to record various country blues musicians. "He brought his stuff down and recorded me right in my house," Muddy recalled in Rolling Stone, "and when he played back the first song I sounded just like anybody's records. Man, you don't know how I felt that Saturday afternoon when I heard that voice and it was my own voice. Later on he sent me two copies of the pressing and a check for twenty bucks, and I carried that record up to the corner and put it on the jukebox. Just played it and played it and said, `I can do it, I can do it.'" Lomax came back again in July 1942 to record Muddy again. Both sessions were eventually released as Down On Stovall's Plantation on the Testament label.

In 1943, Muddy headed back to Chicago with the hope of becoming a full-time professional musician. He lived with a relative for a short period while driving a truck and working in a factory by day and performing at night. Big Bill Broonzy, one of the leading bluesmen in Chicago at the time, helped Muddy break into the very competitive market by allowing him to open for his shows in the rowdy clubs. In 1945, his uncle Joe Grant gave him his first electric guitar which enabled him to be heard above the noisy crowds.

In 1946, he recorded some tunes for Mayo Williams at Columbia but they weren't released at the time. Later that year he began recording for Aristocrat, a newly-formed label run by two brothers, Leonard and Phil Chess. In 1947, he played guitar with Sunnyland Slim on piano on the cuts "Gypsy Woman" and "Little Anna Mae." These were also shelved, but in 1948 "I Can't Be Satisfied" and "I Feel Like Going Home" became big hits and his popularity in clubs began to take off. Soon after, Aristocrat changed their label name to Chess Records and Muddy's signature tune "Rollin' Stone" also became a smash hit. Initially, the Chess brothers would not allow Muddy to use his own musicians in the recording studio; instead he was provided with a backing bass by Ernest "Big" Crawford, or by musicians assembled specifically for the recording session, including "Baby Face" Leroy Foster and Johnny Jones. Gradually Chess relented, and by September 1953 he was recording with arguably the best blues group ever: Little Walter Jacobs on harmonica; Jimmy Rogers on guitar; Elga Edmonds (a.k.a. Elgin Evans) on drums; Otis Spann on piano. The band recorded a series of blues classics during the early 1950s, some with the help of bassist/songwriter Willie Dixon, including "Hoochie Coochie Man" (Number 8 on the R&B charts), "I Just Want to Make Love to You" (Number 4), and "I'm Ready". 

Muddy, along with his former harmonica player Little Walter Jacobs and recent southern transplant Howlin' Wolf, reigned over the early 1950s Chicago blues scene, his band becoming a proving ground for some of the city's best blues talent. While Little Walter continued a collaborative relationship long after he left Muddy's band in 1952, appearing on most of Muddy's classic recordings throughout the 1950s, Muddy developed a long-running but generally good-natured rivalry with Wolf. Wolf's band, like Muddy's, featured an all-star lineup, including the now-legendary guitarist Hubert Sumlin. Wolf also competed with Waters for the songwriting attention of Willie Dixon and recorded a number of Dixon tunes. The success of Muddy's ensemble paved the way for others in his group to break away and enjoy their own solo careers. In 1952 Little Walter left when his single "Juke" became a hit, and in 1955 Rogers quit to work exclusively with his own band, which had been a sideline until that time. Although he continued working with Muddy's band, Otis Spann enjoyed a solo career and many releases under his own name beginning in the mid-1950s.

Muddy headed to England in 1958 and shocked audiences (whose only previous exposure to blues had come via the acoustic folk/blues sounds of acts such as Sonny Terry & Brownie McGhee and Big Bill Broonzy) with his loud, amplified electric guitar and thunderous beat. His performance at the 1960 Newport Jazz Festival, recorded and released as his first live album, At Newport 1960, helped turn on a whole new generation to Waters' sound. He expressed dismay when he realized that members of his own race were turning their backs on the genre while a white audience had shown increasing respect for the blues. However, for the better part of twenty years (since his last big hit in 1956, "I'm Ready") Muddy was put on the back shelf by the Chess label and recorded albums with various "popular" themes: Brass And The Blues, Electric Mud, etc. In 1967, he joined forces with Bo Diddley, Little Walter and Howlin' Wolf to record the Super Blues and The Super Super Blues Band pair of albums of Chess blues standards. In 1972 he went back to England to record The London Muddy Waters Sessions with Rory Gallagher, Steve Winwood, Rick Grech and Mitch Mitchell  —  but their playing was not up to his standards. "These boys are top musicians, they can play with me, put the book before 'em and play it, you know," he told Guralnick. "But that ain't what I need to sell my people, it ain't the Muddy Waters sound. An' if you change my sound, then you gonna change the whole man."

Muddy's long-time wife Geneva died of cancer on March 15, 1973. A devastated Muddy was taken to a doctor and told to quit smoking, which he did. Gaining custody of some of his "outside kids", he moved them into his home, eventually buying a new house in suburban, all-white Westmont. Another teenage daughter turned up while on tour in New Orleans; Big Bill Morganfield was introduced to his Dad after a gig in Florida. Florida was also where Muddy met his future wife, the 19-year-old Marva Jean Brooks whom he nicknamed "Sunshine".

On November 25, 1976, Muddy Waters performed at The Band's farewell concert at Winterland in San Francisco. The concert was released as both a record and a film, The Last Waltz, featuring a performance of "Mannish Boy" with Paul Butterfield on harmonica.

In 1977 Johnny Winter convinced his label, Blue Sky, to sign Muddy, the beginning of a fruitful partnership. His "comeback" LP, Hard Again, was recorded in just two days and was a return to the original Chicago sound he had created 25 years earlier, thanks to Winter's production. Former sideman James Cotton contributed harmonica on the Grammy Award-winning album and a brief but well-received tour followed. The Muddy Waters Blues Band at the time included guitarists Bob Margolin and Luther Johnson, pianist Pinetop Perkins, harmonica player Jerry Portnoy, bassist Calvin "Fuzz" Jones and drummer Willie "Big Eyes" Smith. On "Hard Again", Winter played guitar in addition to producing; Muddy asked James Cotton to play harp on the session, and Cotton brought his own bassist Charles Calmese. According to Margolin's liner notes, Muddy did not play guitar during these sessions. Hard Again was the first studio collaboration between Waters and Winter, who produced his final four albums, the others being I'm Ready, King Bee, and Muddy "Mississippi" Waters - Live, for Blue Sky, a Columbia Records subsidiary.

In 1978 Winter recruited two of Muddy's cohorts from the early '50s, Big Walter Horton and Jimmy Rogers, and brought in the rest of his touring band at the time (harmonica player Jerry Portnoy, guitarist Luther "Guitar Junior" Johnson, and bassist Calvin Jones) to record Waters' I'm Ready LP, which came close to the critical and commercial success of Hard Again. The comeback continued in 1979 with the lauded LP Muddy "Mississippi" Waters Live. On the album, Muddy is accompanied by his touring band, augmented by Johnny Winter on guitar. The set list contains most of his biggest hits, and the album has an energetic feel. King Bee the following year concluded Waters' reign at Blue Sky, and these last four LPs turned out to be his biggest-selling albums ever. King Bee was the last album Muddy Waters recorded. During the sessions for King Bee, Waters, his manager, and his band were involved in a dispute over money. According to the liner notes by Bob Margolin, the conflict arose from Waters' health being on the wane and consequently playing fewer engagements. The band members wanted more money for each of the fewer gigs they did play in order to make ends meet. Ultimately a split occurred and the entire band quit. Because of the tensions in the studio preceding the split, Winter felt the sessions had not produced enough solid material to yield an entire album. He subsequently filled out King Bee with outtakes from earlier Blue Sky sessions and the cover photograph was by David Michael Kennedy. For the listener, King Bee is a leaner and meaner record. Less of the good-time exuberance present on the previous two outings is present here. 

In 1981, Waters was invited to perform at Chicago Fest, the city's top outdoor music festival. He was joined onstage by Johnny Winter — who had successfully produced Waters’ most recent albums — and played classics like “Mannish Boy,” “Trouble No More” and “Mojo Working” to a new generation of fans. This historic performance was made available on DVD in 2009 by Shout! Factory.

In 1982, declining health dramatically curtailed Muddy's performance schedule. Muddy Waters' last public performance took place when he sat in with Eric Clapton's band at a Clapton concert in Florida in autumn of 1982.

On April 30, 1983 Muddy Waters died in his sleep, at his home in Westmont, Illinois. At his funeral at Restvale Cemetery in Alsip, Illinois, throngs of blues musicians and fans showed up to pay tribute to one of the true originals of the art form. "Muddy was a master of just the right notes," John P. Hammond, told Guitar World magazine. "It was profound guitar playing, deep and simple... more country blues transposed to the electric guitar, the kind of playing that enhanced the lyrics, gave profundity to the words themselves." Following Waters' death, B.B. King told Guitar World, "It's going to be years and years before most people realize how greatly he contributed to American music".