The middle of three brothers, House was born in Riverton, two miles from Clarksdale, Mississippi. Around age seven or eight, he was brought by his mother to Tallulah, Louisiana, after his parents separated. The young Son House was determined to become a Baptist preacher, and at age 15 began his preaching career. Despite the church's firm stand against blues music and the sinful world which revolved around it, House became attracted to it and taught himself guitar in his mid 20s, after moving back to the Clarksdale area, inspired by the work of Willie Wilson. He began playing alongside Charley Patton, Willie Brown, Robert Johnson and Fiddlin’ Joe Martin around Robinsonville, Mississippi, and north to Memphis, Tennessee, until 1942.

After killing a man, allegedly in self-defense, he spent time at the Mississippi State Penitentiary (Parchman Farm) in 1928 and 1929. The official story on the killing is that sometime around 1927 or 1928, he was playing in a juke joint when a man went on a shooting spree. Son was wounded in the leg, and shot the man dead. He received a 15-year sentence at Parchman Farm prison.

Son House recorded for Paramount Records in 1930 and for Alan Lomax from the Library of Congress in 1941 and 1942. He then faded from public view until the country blues revival in the 1960s when, after a long search of the Mississippi Delta region by Nick Perls, Dick Waterman and Phil Spiro, he was "re-discovered" in June 1964 in Rochester, New York, where he had lived since 1943. House had been retired from the music business for many years, working for the New York Central Railroad, and was completely unaware of the international revival of enthusiasm for his early recordings.

He subsequently toured extensively in the US and Europe and recorded for CBS records. Like Mississippi John Hurt, he was welcomed into the music scene of the 1960s and played at the Newport Folk Festival in 1964, the New York Folk Festival in July 1965, and the October 1967 European tour of the American Folk Festival along with Skip James and Bukka White.

Son House can be seen in the documentary The Howling Wolf Story. House and Howlin' Wolf had been close early in Wolf's career. However, in the documentary, when Wolf was performing during the 1966 Newport Festival, House was drunk and making a lot of noise during Wolf's set. This angered Wolf who started telling House, from the stage, that all he cared about was whiskey and that he had had a chance to do something with his life but threw it away, to paraphrase Wolf.

The young guitarist Alan Wilson (Canned Heat) was one of Son House's biggest fans. The producer John Hammond Sr. asked Alan Wilson, who was just 22 years old, to teach "Son House how to play like Son House," because Alan Wilson had such a good knowledge of the blues styles. The album The Father of Delta Blues - The Complete 1965 Sessions was the result. Son House played with Alan Wilson live. It can be heard on the album John - the Revelator: The 1970 London Sessions.

In the summer of 1970, House toured Europe once again, including an appearance at the Montreux Jazz Festival; a recording of his London concerts was released by Liberty Records.

Ill health plagued his later years and in 1974 he retired once again, and later moved to Detroit, Michigan, where he remained until his death from cancer of the larynx. He was buried at the Mt. Hazel Cemetery. Members of the Detroit Blues Society raised money through benefit concerts to put a fitting monument on his grave. He had been married five times.

House's innovative style featured strong, repetitive rhythms, often played with the aid of a bottleneck, coupled with singing that owed more than a nod to the field hollers of the chain gangs. Describing House's 1967 appearance at the De Montfort Hall in Leicester, England, Bob Groom wrote in Blues World magazine:

It is difficult to describe the transformation that took place as this smiling, friendly man hunched over his guitar and launched himself, bodily it seemed, into his music. The blues possessed him like a 'lowdown shaking chill' and the spellbound audience saw the very incarnation of the blues as, head thrown back, he hollered and groaned the disturbing lyrics and flailed the guitar, snapping the strings back against the fingerboard to accentuate the agonized rhythm. Son's music is the centre of the blues experience and when he performs it is a corporeal thing, audience and singer become as one.

House was the primary influence on Muddy Waters and also an important influence on Robert Johnson. It was House who, speaking to awe-struck young blues fans in the 1960s, spread the legend that Johnson had sold his soul to the Devil in exchange for his musical powers.

More recently, House's music has influenced the blues-rock group The White Stripes, who covered his song "Death Letter" (also reworked by Skip James and Robert Johnson) on their album De Stijl, and later performed it at the 2004 Grammy Awards. The version on De Stijl contains five of the verses from the Son House original. The eighth verse (one of the ones that was left off) was added to the song "Dead Leaves And The Dirty Ground" on their third album White Blood Cells.

The White Stripes incorporated sections of a traditional song Son House recorded — "John the Revelator" — into the song "Cannon" from their eponymous debut album The White Stripes. Jack White of The White Stripes has cited House's a cappella song, "Grinnin' in Your Face", as his favorite song.

Another musician deeply influenced by Son House is the slide player John Mooney, who in his teens learned slide guitar from Son House while House was living in Rochester, New York.

Several of House's songs were recently featured in the motion picture soundtrack of Black Snake Moan (2006).