Jean's death in 1736 set his son free to choose his own career, and in 1741 he graduated in medicine at Reims and returned to his hometown, planning to practice as a physician.

At about this time, Georges-Louis Leclerc de Buffon, also a native of Montbard, was preparing to bring out a multi-volume work on natural history, the Histoire naturelle, générale et particulière, and in 1742 he invited Daubenton to assist him by providing anatomical descriptions.

In 1744, Daubenton became a member of the French Academy of Sciences as an adjunct botanist, and Buffon appointed him keeper and demonstrator of the king's cabinet in the Jardin du Roi.

In the first section of the Histoire naturelle, Daubenton gave descriptions and details of the dissection of 182 species of quadrupeds, thus securing himself a high reputation as a comparative anatomist.

In December 1799 he was appointed a member of the senate, but at the first meeting which he attended he fell from his seat in an apoplectic fit and, after a short illness, died at Paris.