As a child he was a choirboy at St. Nicholas in Mons and because of his beautiful voice was kidnapped three times for other choirs. He was taken into the service of Ferdinand of Gonzaga, general to Charles V, and travelled with the imperial army in its French campaign in 1544. He accompanied Gonzaga to Italy in 1544, where he remained for 10 years. From 1553 to 1554 he was chapelmaster of the papal church of St. John Lateran at Rome, a post later held by Palestrina. Following a sojourn in Antwerp (1555–56), he joined the court chapel of Duke Albrecht V of Bavaria in Munich, where, except for some incidental journeys, he remained for the rest of his life. In 1570 the Emperor Maximilian raised him to the nobility; and, when Lasso dedicated a collection of his masses (1574) to Pope Gregory XIII, he received the knighthood of the Golden Spur.

Of Lasso's more than 2,000 compositions, many appeared in print between 1555, when his first book of Italian madrigals was published in Venice, and 1604, when a posthumous collection of 516 Latin motets (motet) (religious choral works), Magnum Opus Musicum, was published by his sons. Certain volumes stand out as landmarks in his career: his first collection of motets (1556) established his mastery in a field to which he contributed all his life; a comprehensive anthology of his chansons (chanson), or French part-songs (1570), helped to consolidate his position as the leading composer in this genre. In addition to his madrigals (Italian choral pieces) and chansons, he published seven collections of lieder (German part-songs). Probably his best known work is his sombre, impressive collection of penitential psalms, Psalmi Davidis Poenitentiales (1584). Its rediscovery and edition in 1838 by S.W. Dehn initiated a revival of interest in Lasso's works.

Lasso was a master in the field of sacred music and was equally at home in secular composition. In the latter field his internationalism is striking, encompassing Italian, French, and German genres. His religious works have a particular emotional intensity. He took great care to mirror the meaning of his texts in his music, a trait that looked forward to the Baroque style of the early 17th century.