Henslowe was born in Lindfield, Sussex, England. By 1577, he was living in Southwark, London, and around that time he married a wealthy widow, which allowed him to invest in property and a diverse range of industries. On top of his many business interests, he was a churchwarden and held minor positions at court.
In 1587, Henslowe and a partner built The Rose, which Henslowe also managed. Later he built the Hope and Fortune theatres. He also had interests in the Newington Butts Theatre and The Swan. His most important theatrical partnership was with Edward Alleyn, the popular lead actor of the Admiral's Men, who married his stepdaughter, Joan.
Henslowe's Diary is a valuable source of information on the theatrical history of the period. Rather than being a diary in the modern sense, it was really a notebook in which he made records of payments to writers, box office takings, and various other theatrical dealings. Also of interest are records of the purchase of expensive costumes and of stage properties, like the dragon in Christopher Marlowe's Doctor Faustus, which give an insight into the staging of plays in the Elizabethan theatre.
Henslowe recorded payments to most of the dramatists of the day, including Ben Jonson, Thomas Middleton, George Chapman, Thomas Dekker, John Marston and Michael Drayton. Plays with Shakespearean titles like, Hamlet, Henry VI, Henry V, Taming of the Shrew and others, are found in the diary; and, in 1599, Henslowe paid Dekker and Henry Chettle for a play called Troilus and Cressida. However, there is no mention of William Shakespeare in Henslowe's diary (despite the efforts of John Payne Collier).
Henslowe died in London. His papers are held at Dulwich College, which was founded by Alleyn.