His father, Edmond Pelouze, was an industrial chemist and the author of several technical handbooks. The son, after spending some time in a pharmacy at La Fère acted as laboratory assistant to Gay-Lussac and Jean Louis Lassaigne at Paris from 1827 to 1829. In 1830 he was appointed associate professor of chemistry at Lille, but returning to Paris next year became repetiteur, and subsequently professor at the École polytechnique. He also held the chair of chemistry at the Collège de France, and in 1833 became assayer to the mint and in 1848 president of the Commission des Monnaies. He resigned all his public positions in 1852.
After the coup d'état in 1851 he resigned his appointments, but continued to conduct an experimental laboratory-school he had started in 1846. There he worked with the explosive material guncotton and other nitrosulphates. His student Ascanio Sobrero was the discoverer of nitroglycerin (1847), and another student, Alfred Nobel, was to take that discovery on to great heights in the form of commercial explosives including dynamite. He was a major inspiration for both students.
Though Pelouze made no discovery of outstanding importance, he was a busy investigator, his work including researches on salicin, on beetroot sugar, on various organic acids (gallic, malic, tartaric, butyric, lactic, etc.), on oenanthic ether (with Liebig), on the nitrosulphates, on guncotton, and on the composition and manufacture of glass.
He also carried out determinations of the atomic weights of several elements, and with E. Fremy, published Traité de chimie générale (1847–1850); Abrégé de chimie (1848); and Notions générales de chimie (1853).
He and his wife Marguerite lived in the famous French castle Château de Chenonceau from 1864 until his death in 1867; she continued to inhabit it until at least 1878.