He was born in Dublin, the great-grandson of Josiah Hort, Archbishop of Tuam in the eighteenth century. In 1846 he passed from Rugby School to Trinity College, Cambridge, where he was the contemporary of EW Benson, BF Westcott and JB Lightfoot. The four men became lifelong friends and fellow-workers. In 1850 Hort took his degree, being third in the classical tripos, and in 1852 he became fellow of his college. In 1854, in conjunction with JEB Mayor and Lightfoot, he established the Journal of Classical and Sacred Philology, and plunged eagerly into theological and patristic study. He had been brought up in the strictest principles of the Evangelical school, but at Rugby he fell under the influence of Thomas Arnold and Archibald Campbell Tait, and his acquaintance with John Frederick Denison Maurice and Charles Kingsley finally gave his opinions a direction towards Liberalism.
In 1857 he was married, and accepted the college living of St Ippolyts, near Hitchin, in Hertfordshire, where he remained for fifteen years. During his time there he took part in discussions on university reform, continued his studies, and wrote essays for various periodicals. In 1870 he was appointed a member of the committee for revising the translation of the New Testament, and in 1871 he delivered the Hulsean lectures before the university. Their title was The Way, the Truth, and the Life, but they were not prepared for publication until many years after their delivery. In 1872 he accepted a fellowship and lectureship at Emmanuel College, Cambridge; in 1878 he was made Hulsean professor of divinity, and in 1887 Lady Margaret's Professor of Divinity.
In 1881 he published, with his friend Westcott, an edition of the text of the New Testament based on their text critical work. The Revision Committee had largely accepted this text, even before its publication, as a basis for their translation of the New Testament. Its appearance created a sensation among scholars, and it was attacked in many quarters, but on the whole it was received as being much the nearest approximation yet made to the original text of the New Testament. The introduction was the work of Hort. His first principle was, "Knowledge of Documents should precede Final Judgments upon Readings".
Hort died on 30 November 1892, worn out by intense mental labour. Next to his Greek Testament his best-known work is The Christian Ecclesia (1897). Other publications are: Judaistic Christianity (1894); Village Sermons (two series); Cambridge and other Sermons; Prolegomena to ... Romans and Ephesians (1895); The Ante-Nicene Fathers (1895); and two Dissertations, on the reading of a Greek word in John i.18, and on The Constantinopolitan and other Eastern Creeds in the Fourth Century. All are models of exact scholarship and skilful use of materials. His Life and Letters was edited by his son, Sir Arthur Hort, Bart. (1896).