Regnault came from a poor background in Aachen (now in Germany) and started work as a draper's assistant. He entered the Ecole Polytechnique in 1830 and later worked under Justus von Liebig at Giessen. He was successively professor of chemistry at the University of Lyons and the Ecole Polytechnique (1840). He became professor of physics at the Collège de France (1841) and finally director of the Sèvres porcelain factory in 1854.

Regnault's main work was in physics on the properties of gases and in particular the more accurate determination of many physical and chemical effects. Through his meticulous studies he showed, for example, that the law of Pierre Dulong and Alexis Petit was only approximately true when pure samples were taken and temperatures carefully measured. He also worked on the properties of gases – Joseph Gay-Lussac had claimed that a gas will increase by 1/266 of its volume for each increase of temperature of 1°C but Regnault showed that the true increase was 1/273. In addition he made accurate measurements of specific and latent heats and reliable determinations of atomic weights. Regnault is credited with the invention of the air thermometer.

In chemistry, Regnault discovered various organic chlorides that have since become important industrially, including vinyl chloride and carbon tetrachloride. He also took samples of air from different parts of the world and demonstrated that wherever it comes from it contains about 21% oxygen.