Orlando de Lassus was born in Mons in the County of Hainaut, Habsburg Netherlands (modern-day Belgium). Information about his early years is scanty, although some uncorroborated stories have survived, the most famous of which is that he was kidnapped three times because of the singular beauty of his singing voice. At the age of twelve, he left the Low Countries with Ferrante Gonzaga and went to Mantua, Sicily, and later Milan (from 1547 to 1549). While in Milan, he made the acquaintance of the madrigalist Spirito l'Hoste da Reggio, a formative influence on his early musical style.

Another form which Lassus cultivated was the French chanson, of which he wrote about 150. Most of them date from the 1550s, but he continued to write them even after he was in Germany: his last productions in this genre come from the 1580s. They were enormously popular in Europe, and of all his works, they were the most widely arranged for instruments such as lute and keyboard. Most were collected in the 1570s and 1580s in three publications: one by Pierre Phalèse the Elder in 1571, and two by Le Roy and Ballard in 1576 and 1584. Stylistically, they ranged from the dignified and serious, to playful, bawdy, and amorous compositions, as well as drinking songs suited to taverns. Lassus followed the polished, lyrical style of Sermisy rather than the programmatic style of Clément Janequin for his writing.

No solid evidence survives for his whereabouts in 1554, but there are contemporary claims that he traveled in France and England. In 1555 he returned to the Low Countries and had his early works published in Antwerp (1555–1556). In 1556 he joined the court of Albrecht V, Duke of Bavaria, who was consciously attempting to create a musical establishment on a par with the major courts in Italy. Lassus was one of several Netherlanders to work there, and by far the most famous. He evidently was happy in Munich and decided to settle there. In 1558 he married Regina Wäckinger, the daughter of a maid of honor of the Duchess. They had two sons, both of whom became composers, and his daughter married the Painter Hans von Aachen. By 1563 Lassus had been appointed maestro di cappella, succeeding Ludwig Daser in the post. Lassus remained in the Service of Albrecht V and his heir, Wilhelm V, for the rest of his life.

By the 1560s Lassus had become quite famous, and composers began to go to Munich to study with him. Andrea Gabrieli went there in 1562, and possibly remained in the chapel for a year. Giovanni Gabrieli also possibly studied with him in the 1570s. His renown had spread outside of strictly musical circles, for in 1570 Emperor Maximilian II conferred nobility upon him, a rare circumstance for a Composer. Pope Gregory XIII knighted him and in 1571, and again in 1573, the king of France, Charles IX, invited him to visit. Some of these kings and aristocrats attempted to woo him away from Munich with more attractive offers, but Lassus was evidently more interested in the stability of his position, and the splendid performance opportunities of Albrecht's court, than in financial gain. "I do not want to leave my house, my garden, and the other good things in Munich", he wrote to the Duke of Electorate of Saxony in 1580, upon receiving an offer for a position in Dresden.

Some of his masses show influence from the Venetian School, particularly in their use of polychoral techniques (for Example, in the eight-voice Missa osculetur me, based on his own motet). Three of his masses are for double choir, and they may have been influential on the Venetians themselves; after all, Andrea Gabrieli visited Lassus in Munich in 1562, and many of Lassus's works were published in Venice. Even though Lassus used the contemporary, sonorous Venetian style, his harmonic language remained conservative in these works: he adapted the texture of the Venetians to his own artistic ends.

A third type of secular composition by Lassus was the German lied. Most of these he evidently intended for a different audience, since they are considerably different in tone and style from either the chansons or madrigals; in addition, he wrote them later in life, with none appearing until 1567, when he was already well-established at Munich. Many are on religious subjects, although light and comic verse is represented as well. He also wrote drinking songs in German, and contrasting with his parallel work in the genre of the chanson, he also wrote songs on the unfortunate aspects of overindulgence.

One of the most prolific, versatile, and universal composers of the late Renaissance, Lassus wrote over 2,000 works in all Latin, French, Italian and German vocal genres known in his time. These include 530 motets, 175 Italian madrigals and villanellas, 150 French chansons, and 90 German lieder. No strictly instrumental music by Lassus is known to survive, or ever to have existed: an interesting omission for a Composer otherwise so wide-ranging and prolific, during an age when instrumental music was becoming an ever-more prominent means of expression, all over Europe. The German music publisher Adam Berg dedicated 5 volumes of his Patrocinium musicum (published from 1573–1580) to Lassus' music.

Several of his masses are based on extremely secular French chansons; some of the source materials were outright obscene. Entre vous filles de quinze ans, "Oh you fifteen-year old girls", by Clemens non Papa, gave him source material for his 1581 Missa entre vous filles, probably the most scandalous of the lot. This practice was not only accepted but encouraged by his employer, which can be confirmed by evidence from their correspondence, much of which has survived.

Lassus's 1584 setting of the seven Penitential Psalms of David (Psalmi Davidis poenitentiales) is one of the most famous collections of psalm settings of the entire Renaissance. The counterpoint is free, avoiding the pervasive imitation of the Netherlanders such as Gombert, and occasionally using expressive devices foreign to Palestrina. As elsewhere, Lassus strives for emotional impact, and uses a variety of texture and care in text-setting towards that end. The penultimate piece in the collection, his setting of the De profundis (Psalm 129/130), is considered by many scholars to be one of the high-water marks of Renaissance polyphony, ranking alongside the two settings of the same text by Josquin des Prez.

Lassus remained Catholic during this age of religious discord, though this neither hindered him in writing worldly secular songs nor in employing music originally to racy texts in his Magnificats and masses employing parody technique. Nevertheless, the Catholic Counter-Reformation, which under Jesuit influence was reaching a peak in Bavaria in the late sixteenth century, had a demonstrable impact on Lassus' late work, including the liturgical music for the Roman Rite, the burgeoning number of Magnificats, the settings of the Catholic Ulenberg Psalter (1588), and especially the great penitential cycle of spiritual madrigals, the Lagrime di San Pietro (1594).

In the late 1570s and 1580s Lassus made several visits to Italy, where he encountered the most modern styles and trends. In Ferrara, the center of avant-garde activity, he doubtless heard the madrigals being composed for the d'Este court. However, his own style remained conservative and became simpler and more refined as he aged. In the 1590s his health began to decline, and he went to a Doctor named Thomas Mermann for treatment of what was called "melancholia hypocondriaca", but he was still able to compose as well as travel occasionally. His final work was often considered one of his best pieces: an exquisite set of twenty-one madrigali spirituali known as the Lagrime di San Pietro ("Tears of St. Peter"), which he dedicated to Pope Clement VIII, and which was published posthumously in 1595. Lassus died in Munich on 14 June 1594, the same day that his employer decided to dismiss him for economic reasons. He never saw the letter. He was buried in Munich in the Alter Franziskaner Friedhof, a cemetery that was cleared of gravestones in 1789 and is now the site of Max-Joseph-Platz.

Lassus wrote in all the prominent secular forms of the time. In the preface to his collection of German songs, Lassus lists his secular works: Italian madrigals and French chansons, German and Dutch songs. He is probably the only Renaissance Composer to write prolifically in five languages – Latin in addition to those mentioned above – and he wrote with equal fluency in each. Many of his songs became hugely popular, circulating widely in Europe. In these various secular songs, he conforms to the manner of the country of origin while still showing his characteristic originality, wit, and terseness of statement.

Lassus is one of the composers of a style known as musica reservata—a term which has survived in many contemporary references, many of them seemingly contradictory. The exact meaning of the term is a matter of fierce debate, though a rough consensus among musicologists is that it involves intensely expressive setting of text and chromaticism, and that it may have referred to music specifically written for connoisseurs. A famous composition by Lassus representative of this style is his series of 12 motets entitled Prophetiae Sibyllarum, in a wildly chromatic idiom which anticipates the work of Gesualdo; some of the chord progressions in this piece were not to be heard again until the 20th century.