Fletcher was born in December 1579 (baptised December 20) in Rye, Sussex, and died of the plague in August 1625 (buried August 29 in St. Saviour's, Southwark). His father Richard Fletcher was an ambitious and successful cleric who was in turn Dean of Peterborough, Bishop of Bristol, Bishop of Worcester, and Bishop of London (shortly before his death) as well as chaplain to Queen Elizabeth. As dean of Peterborough, Richard Fletcher, at the execution of Mary, Queen of Scots, at Fotheringay "knelt down on the scaffold steps and started to pray out loud and at length, in a prolonged and rhetorical style as though determined to force his way into the pages of history" and who cried out at her death, "So perish all the Queen's enemies!" Richard Fletcher died shortly after falling out of favor with the queen, over a marriage the queen had advised against. He appears to have been partly rehabilitated before his death in 1596; however, Fletcher died substantially in debt. The upbringing of John Fletcher and his seven siblings was entrusted to his paternal uncle Giles Fletcher, a poet and minor official. His uncle's connections ceased to be a benefit, and may even have become a liability, after the rebellion of the Earl of Essex, who had patronized him.

Fletcher appears to have entered Corpus Christi College, Cambridge University, in 1591, at the age of eleven. It is not certain that he took a degree, but evidence suggests that he was preparing for a career in the church. Little is known about his time at college, but he evidently followed the same path previously trod by the University wits before him, from Cambridge to the burgeoning commercial theater of London. In 1606, he began to appear as an author for the Children of the Queen's Revels, then performing at the Blackfriars Theatre. Commendatory verses by Richard Brome in the Beaumont and Fletcher 1647 folio place Fletcher in the company of Ben Jonson; a comment of Jonson's to Drummond corroborates this claim, although it is not known when this friendship began. At the beginning of his career, his most important association was with Francis Beaumont. The two wrote together for close to a decade, first for the children and then for the King's Men. According to a legend transmitted or invented by John Aubrey, they also lived together (in Bankside), sharing clothes and having "one wench in the house between them." This domestic arrangement, if it existed, was ended by Beaumont's marriage in 1613, and their dramatic partnership ended after Beaumont fell ill, probably of a stroke, the same year.

By this time, Fletcher had moved into a closer association with the King's Men. He is commonly assumed to have collaborated with Shakespeare on Henry VIII, The Two Noble Kinsmen, and the arguably lost Cardenio, which is probably (according to modern scholarly consensus) the play known as Double Falsehood; a play he wrote singly around this time, The Woman's Prize or the Tamer Tamed, is a sequel to The Taming of the Shrew. In 1616, after Shakespeare's death, Fletcher appears to have entered into an exclusive arrangement with the King's Men similar to that with which Shakespeare had worked; Fletcher wrote only for that company between the death of Shakespeare and his own death nine years later. He never lost his habit of collaboration, working with Nathan Field and later with Philip Massinger, who succeeded him as house playwright for the King's Men. His popularity continued unabated throughout his life; during the winter of 1621, three of his plays were performed at court. He died in 1625, apparently of the plague. He seems to have been buried in what is now Southwark Cathedral, although the precise location is not known; there is a reference by Aston Cockayne to a single grave for Fletcher and Massinger (also buried in Southwark).

His mastery is most notable in two dramatic types, tragicomedy and comedy of manners, both of which exerted a pervasive influence on dramatists in the reign of Charles I and during the Restoration.

Fletcher's early career was marked by one significant failure, of The Faithful Shepherdess, his adaptation of Giovanni Battista Guarini's Il Pastor Fido, which was performed by the Blackfriars Children in 1608. In the preface to the printed edition of his play, Fletcher explained the failure as due to his audience's faulty expectations. They expected a pastoral tragicomedy to feature dances, comedy, and murder, with the shepherds presented in conventional stereotypes — as Fletcher put it, wearing "gray cloaks, with curtailed dogs in strings." Fletcher's preface in defense of his play is best known for its pithy definition of tragicomedy: "A tragicomedy is not so called in respect of mirth and killing, but in respect it wants [i.e., lacks] deaths, which is enough to make it no tragedy; yet brings some near it, which is enough to make it no comedy." A comedy, he went on to say, must be "a representation of familiar people," and the preface is critical of drama which would feature characters whose action violates nature.

In that case, Fletcher appears to have been developing a new style faster than audiences could comprehend. By 1609, however, he had found his stride. With Beaumont, he wrote Philaster, which became a hit for the King's Men and began a profitable connection between Fletcher and that company. Philaster appears also to have initiated a vogue for tragicomedy; Fletcher's influence has been credited with inspiring some features of Shakespeare's late romances (Kirsch, 288 - 90), and his influence on the tragicomic work of other playwrights is even more marked. By the middle of the 1610s, Fletcher's plays had achieved a popularity that rivalled Shakespeare's and which cemented the preeminence of the King's Men in Jacobean London. After Beaumont's retirement and early death in 1616, Fletcher continued working, both singly and in collaboration, until his death in 1625. By that time, he had produced, or had been credited with, close to fifty plays. This body of work remained a major part of the King's Men's repertory until the closing of the theaters in 1642.

During the Commonwealth, many of the playwright's best known scenes were kept alive as drolls, the brief performances devised to satisfy the taste for plays while the theaters were suppressed. At the re-opening of the theaters in 1660, the plays in the Fletcher canon, in original form or revised, were by far the most common fare on the English stage. The most frequently revived plays suggest the developing taste for comedies of manners. Among the tragedies, The Maid's Tragedy and, especially, Rollo Duke of Normandy held the stage. Four tragicomedies (A King and No King, The Humorous Lieutenant, Philaster, and The Island Princess) were popular, perhaps in part for their similarity to and foreshadowing of heroic drama. Four comedies (Rule a Wife And Have a Wife, The Chances, Beggars' Bush, and especially The Scornful Lady) were also popular. Yet the popularity of these plays relative to those of Shakespeare and to new productions steadily eroded. By around 1710, Shakespeare's plays were more frequently performed, and the rest of the century saw a steady erosion in performance of Fletcher's plays. By 1784, Thomas Davies asserted that only Rule a Wife and The Chances were still current on stage; a generation later, Alexander Dyce mentioned only The Chances. Since then Fletcher has increasingly become a subject only for occasional revivals and for specialists. Fletcher and his collaborators have been the subject of important bibliographic and critical studies, but the plays have been revived only infrequently.

Fletcher's canon presents unusual difficulties of attribution. He collaborated regularly and widely, most often with Beaumont and Massinger but also with Nathan Field, Shakespeare and others. Some of his early collaborations with Beaumont were later revised by Massinger, adding another layer of complexity to unravel. Fortunately for scholars and students of English literature, Fletcher also had highly distinctive mannerisms in his creative efforts; his texts reveal a range of peculiarities that effectively identify his presence. He frequently uses ye instead of you, at rates sometimes approaching 50%; he frequently employs 'em for them, along with a set of other particular preferences in contractions; he adds a sixth stressed syllable to a standard pentameter verse line — most often sir but also too or still or next; he has various other specific habits and preferences. The detection of this pattern, this personal Fletcherian textual profile, has allowed researchers to penetrate the confusions of the Fletcher canon with good success — and has in turn encouraged the use of similar techniques more broadly in the study of literature. Careful bibliography has established the authors of each play with some degree of certainty. Determination of the exact shares of each writer (for instance by Cyrus Hoy) in particular plays is ongoing, based on patterns of textual and linguistic preferences, stylistic grounds, and idiosyncrasies of spelling.