Ordained a priest in 1867, he studied in Rome and in Paris (1871–73), where he was appointed professor at the Catholic Institute (1877–85) and where in 1881 he founded the Bulletin Critique de Littérature, d'Histoire et de Théologie. Persuaded to resign after criticism of his lectures, he taught at the École Supérieure des Lettres from 1885 to 1895, when he was appointed director of the École Française de Rome; he served there until his death. In 1910 he was elected to the French Academy and was made a prothonotary apostolic by Pope Leo XIII.

Duchesne's works include the authoritative edition of the Liber Pontificalis, 2 vol. (1886–92); Autonomies ecclésiastiques: églises séparées (1896; “Ecclesiastical Autonomies: Detached Churches”), dealing with the origin of the Greek and Anglican churches; and Histoire ancienne de l'église chrétienne (Early History of the Christian Church), of which the first three volumes (1905–08) were put on the Index of Forbidden Books, the fourth volume being published posthumously (1925).