He was born in Orléans. A doubtful tradition makes him the illegitimate son of Francis I; but it is evident that he was at least connected with some family of rank and wealth. From Orléans he was taken to Paris about 1521, and after studying under Nicolas Bérauld, the teacher of Coligny, he proceeded in 1526 to Padua. The death of his friend and master, Simon de Villanova, led him, in 1530, to accept the post of secretary to Jean de Langeac, bishop of Limoges and French ambassador to the republic of Venice; he contrived, however, to attend the lectures of the Venetian scholar Battista Egnazio, and found time to write Latin love poems to a Venetian woman named Elena.

Returning to France soon afterwards he proceeded to Toulouse to study law; but there he soon became involved in the violent disputes between the different nations of the university, was thrown into prison, and finally banished by a decree of the parlement. He entered the lists against Erasmus in the famous Ciceronian controversy, in which he took an ultra-Ciceronian stance: In 1535 he published through Sébastien Gryphe at Lyon a Dialogus de imitatione Ciceroniana. The following year saw the appearance of his two folio volumes Commentariorum linguae Latinae. This work was dedicated to Francis I, who gave him the privilege of printing during ten years any works in Latin, Greek, Italian or French, which were the product of his own pen or had received his supervision; and accordingly, on his release from an imprisonment occasioned by his homicide of a painter named Compaing, he began at Lyon his typographical and editorial labours.

He endeavoured to conciliate his opponents by publishing a Cato christianus, in which he made profession of his creed. The catholicity of his literary appreciation, was soon displayed by the works which proceeded from his press: ancient and modern, sacred and secular, from the New Testament in Latin to Rabelais in French. But before the term of his privilege expired his labours were interrupted by his enemies, who succeeded in imprisoning him (1542) on the charge of atheism.

After imprisonment for fifteen months, Dolet was released by the advocacy of Pierre Duchatel, Bishop of Tulle. He escaped from a second imprisonment (1544) by his own ingenuity, but, venturing back from Piedmont, whence he had fled in order to print at Lyon the letters by which he appealed for justice to the king of France, the queen of Navarre and the parlement of Paris, he was again arrested, and branded as a relapsed atheist by the theological faculty of the Sorbonne. On 3 August 1546 (his 37th birthday), he was strangled and burned in the Place Maubert. On his way there he is said to have composed the punning pentameter Non dolet ipse Dolet, sed pia turba dolet.

Whether Dolet is to be classed with the representatives of Protestantism or with the advocates of anti-Christian rationalism has been frequently disputed; by the principal Protestants of his own time he was not recognized, and by Calvin he was formally condemned, along with Agrippa and his master Villanova, as having uttered execrable blasphemies against the Son of God; but, to judge by the religious character of a large number of the books which he translated or published, these condemnations seem altogether misplaced. His repeated advocacy of the reading of the Scriptures in the vernacular tongue is especially noticeable. Dolet has been referred to as an Anti-Trinitarian, whether this is correct or not needs verification.

The trial of Dolet was published (1836) by A.H. Taillandier from the registers of the parlement of Paris. A statue of Dolet was erected on the Place Maubert in Paris in 1889.