A member of a distinguished intellectual family, Stephen was educated at Eton, at King's College, London, and at Trinity Hall, Cambridge, where he was elected to a fellowship in 1854 and became junior tutor in 1856. He was ordained in 1859, but his philosophical studies, combined probably with the controversy that followed the publication of Charles Darwin's Origin of Species (1859), caused him to lose his faith; in 1862 he resigned his tutorship and two years later left Cambridge to live in London.

Through his brother, James Fitzjames Stephen, a contributor to the Saturday Review, Stephen gained entry to the literary world, contributing to many periodicals. From 1871 to 1882 he edited The Cornhill Magazine, for which he wrote literary criticism (republished in the three series of Hours in a Library, 1874–79). Stephen was one of the first serious critics of the novel. Thomas Hardy, Robert Louis Stevenson, Edmund Gosse, and Henry James were among those whom Stephen, as an editor, encouraged.

His greatest learned work was History of English Thought in the Eighteenth Century (1876). His philosophical study The English Utilitarians (1900) was somewhat less successful, though it is still a useful source. His philosophical contribution to the rationalist tradition, Science of Ethics (1882), attempted to wed evolutionary theory to ethics, and An Agnostic's Apology appeared in 1893. Stephen's most enduring legacy, however, is the Dictionary of National Biography, which he edited from 1882 to 1891; he edited the first 26 volumes and contributed 378 biographies. In recognition of this service to letters he was knighted in 1902. Stephen's English Literature and Society in the Eighteenth Century (1904) was a pioneer work in the sociological study of literature.

Stephen was shy and given to silence, the more so after the death in 1875 of his first wife, Harriet Marian (“Minny”), the second daughter of William Makepeace Thackeray. In 1878 he married Julia Jackson, a widow, and among their four children were the painter Vanessa Bell and the novelist Virginia Woolf (Woolf, Virginia).