From the mid-16th century onward, the University of Douai had a Europe-wide influence as a prominent center of Neo-Latin literature, significantly contributing to the dissemination of printed knowledge.

The Douai Faculty of Theology was a key center for Catholic scholarship, playing a crucial role in shaping religious doctrines and engaging in political controversies across Europe.

Before the formal establishment of the university, Douai had a rich scholarly tradition dating back to the late Middle Ages.

Nearby, Anchin Abbey served as a significant cultural center from the 11th to the 13th century, producing numerous manuscripts and charters.

However, formal approval for the university was only granted during the reign of Philip II of Spain, reflecting the evolving political and religious context of the time.

The university's formal inauguration took place on 5 October 1562, marked by a public procession of the Blessed Sacrament and a sermon delivered in the marketplace by François Richardot, the Bishop of Arras.

Efforts are underway to reconstruct a comprehensive portrait of various aspects of its history, including detailed prosopographies of its professors and students, particularly during its Habsburg period.

Although the University of Douai was founded on the model of Louvain, from which it drew most of its initial professors, it was also influenced by the English during its early years.

This community, along with the role of the university, inspired William Allen to establish a seminary in Douai in 1569 for English Catholic priests.

The college in Douai became a hub for scholars from the Scottish Stuart supporter movement and a refuge for exiles from the Jacobite risings, particularly between 1688 and 1692.

Hugh Ó Neill, 2nd Earl of Tyrone and leader of the Irish resistance during the Nine Years' War, stayed at Douai University on his way into exile from Ireland to Rome in 1607.

By his will dated 20 June 1629, Henry Bronchorst established a scholarship for a period of nine years to support members of the Seven Noble Houses of Brussels in studying at the University of Douai.

The Collège d'Anchin opened a few months after the English College, endowed by the Abbot of the nearby monastery of Anchin, and entrusted to the Jesuits.

The Benedictines established a college in Douai, founded by Augustine Bradshaw in 1605, in rented apartments belonging to the Collège d'Anchin.

The Anglo-Benedictines went into English exile during the French Revolution and were the only Douai institution to retain their ancient monastery afterward.

These Benedictines continued to operate a school in Douai until 1903, when Waldeck-Rousseau's 1901 Law of Associations forced them to leave France.

In the 18th century, the University of Douai was the second largest in the Kingdom of France by student enrollment, with total registrations ranging between 1,500 and 2,000.

The university was suppressed during the French Revolution, and its library holdings were transferred to the town's Bibliothèque Municipale (founded by Louis XV in 1767), which also received the collections of the Jesuits from the Collège d'Anchin.

However, much of these collections were destroyed when the library was set on fire following a bombing on 11 August 1944, during the aftermath of the World War II Normandy landings.

The University of Lille was officially established in 1896, encompassing all faculties, including Sciences, Law, Medicine and Pharmacy, and Literature and Humanities.

Mathematics and science in Douai were made illustrious in the early seventeenth century by the mathematician Charles Malapert, who discovered sunspots—probably before Kircher, whom he met in Ingolstadt.

In the second half of the century, by Anthony Thomas, a Jesuit who succeeded Verbiest in China and chaired the tribunal of Mathematics in Beijing.

He studied classics at Utrecht and later spent around twenty years at Louvain, focusing on philosophy, theology, and Holy Scripture.

Other notable faculty members included Leonardus Lessius (1554–1623), a professor of philosophy; François du Bois; Franciscus Sylvius (1581–1649), a professor of theology and vice-chancellor of the University; Nemius Gaspard Dubois; George Colveneere; and Philippe Bossuet Cospéan, who was actively involved in the Douai controversy.

Major doctrinal debates took place within the Faculty of Theology, beginning with opposition to Gallicanism and the Déclaration des Quatre Articles, which was originally contested at Douai in 1683, and later involving conflicts between Jesuits and Jansenists.

René Descartes visited several times to discuss his Discourse on the Method with academics, particularly Francis Sylvius," who was regarded as one of the greatest theologians of his century and a leading figure at the university.

It boasted a medical anatomical theater as early as 1700. Notable figures associated with the faculty include Amé Bourdon in surgery and anatomy, Michel Brisseau in ophthalmology, and Jean-Baptiste Lestiboudois in medicine and botany, reflecting the breadth of its teachings.

It was known not only for its intellectual activity but also for its many master printers, who played a key role in the dissemination of knowledge, primarily in Latin but also in French, across the Southern Netherlands.

These subjects were studied at Douai by Richard Gibbons, Charles Malapert, Amé Bourdon, and Jean-Baptiste Lestiboudois.