During the 16th and 17th centuries, the University of Alcalá established itself as one of the most prestigious academic centers in the Hispanic world. It became a focus of knowledge and critical thought, especially in the fields of theology, philosophy, law, classical languages and medicine. Its printing house, the famous Typographia Complutense, was famous for the edition of the Biblia Políglota Complutense, a monumental work coordinated by Cisneros himself.

In 1777, as part of the enlightened reforms of the reign of Charles III, the university was institutionally separated from its emblematic Colegio Mayor and renamed the Real Universidad de Alcalá. This new stage located it in the building previously occupied by the Colegio Máximo de los Jesuitas, also in Alcalá. However, the institution went into a slow decline due to the political, social and economic changes that affected Spain in the late eighteenth and early nineteenth centuries.

Finally, in 1836, the university was moved to Madrid, in a process of centralization of the educational system promoted by the liberal state. It merged with the Royal Studies of San Isidro and the Royal Museum of Natural Sciences to form the Central University of Madrid, the seed of the current Complutense University of Madrid, a direct heir of the Cisnerian tradition.

Throughout its history, the University of Alcalá was the cradle and home of numerous key figures in thought, literature and science. Among its most famous professors and students are Antonio de Nebrija, author of the first grammar of the Castilian language; Saint Thomas of Villanueva; the historian Juan Ginés de Sepúlveda; Saint Ignatius of Loyola, founder of the Society of Jesus; the theologian Domingo de Soto; the chronicler Ambrosio de Morales; Benito Arias Montano, editor of the Biblia Regia; the Jesuit philosopher Francisco Suárez; the Jesuit and historian Juan de Mariana; the physician Francisco Vallés de Covarrubias; the politician Antonio Pérez; mystics such as San Juan de la Cruz; writers of the stature of Mateo Alemán, Lope de Vega, Francisco de Quevedo and Pedro Calderón de la Barca; the enlightened Melchor Gaspar de Jovellanos; the scientist Andrés Manuel del Río, discoverer of vanadium; and the religious Fray Diego Morcillo, among many others.