He was ordained to the priesthood in 1867. Louis Duchesne taught for many years in Saint-Brieuc, then went to study in Paris, where he influenced the reformist Alfred Firmin Loisy, a founder of the failed movement to bring Catholicism into sympathy with science, the modern social sciences and philosophy, called "Catholic modernism" which eventually precipitated the crisis in the Church under Pope Pius X in the years around 1907. In 1876 he became a member of the École française in Rome; he eventually became its director. He was an amateur archaeologist and organized expeditions from Rome to Mount Athos, to Syria, and Asia Minor, from which he gained an interest in the early history of the Roman Catholic Church.
In 1887 he published the results of his thesis, followed by the first complete critical edition of the Liber Pontificalis. (Theodor Mommsen was also working on a critical study, but it was never finished). At a difficult time for critical historians applying modern methods to Church history, drawing together archaeology and topography to supplement literature and setting ecclesiastical events with contexts of social history, Abbé Duchesne was in constant correspondence with like-minded historians among the Bollandists, with their long history of critical editions of hagiographies.
He also wrote Les Sources du martyrologe hyéronimien, Origines du culte chrétien (translated as Christian Worship: Its Origin and Evolution and often reprinted), Fastes épiscopaux de l'ancienne Gaule, and Les Premiers temps de l'État pontifical. These works were universally praised, and he was appointed a commander of the Legion of Honor. However, his Histoire ancienne de l'Église, 1906‑11 (translated as Early History of the Christian Church) was considered too modernist by the Church during the "Modernist crisis" and was placed on the Index of Forbidden Books in 1912.