Vicente Lusitano was born in Olivença, but little else is known for certain about his life, including the dates of his birth and death. Some information is recorded in the 18th century biography by Barbosa Machado: he came from Olivença, became a priest, and was employed as a teacher both at Padua and Viterbo. Machado also called him a mestizo. Very little of what Machado wrote about him has been verified by any other source, except the date of publication (1561) of a music theory treatise at Venice.

As a composer Lusitano wrote a number of choral works, including motets and a madrigal, but he is better known by far for his work as a theorist. In a 1551 debate in Rome, he espoused traditional views on the role of the three genera in music (diatonic, chromatic and enharmonic) over more radical ones put forward by Nicola Vicentino (Lusitano was deemed to have won the debate). His Introdutione facilissima et novissima de canto ferma (Rome, 1553, and again at Venice, 1561), contains an introduction to music, a section on improvised counterpoint, and his views on the three genera.