Born in Aachen in 1810, he moved to Paris following the death of his parents at the age of eight. There, he worked for an upholstery firm until he was eighteen. In 1830, he was admitted to the École Polytechnique, and in 1832 he graduated from the École des mines.

Working under Justus von Liebig at Gießen, Regnault distinguished himself in the nascent field of organic chemistry by synthesizing several chlorinated hydrocarbons (e.g. vinyl chloride, polyvinyl chloride, dichloromethane), and he was appointed professor of chemistry at the University of Lyon. In 1840, he was appointed the chair of chemistry of the École Polytechnique, and in 1841, he became a professor of Physics in the Collège de France.

Beginning in 1843, he began compiling extensive numerical tables on the properties of steam. These were published in 1847, and led to his receiving the Rumford Medal of the Royal Society of London and appointment as Chief Engineer of Mines. In 1851 he was elected a foreign member of the Royal Swedish Academy of Sciences. In 1854 he was appointed director of the porcelain works at Sèvres.

At Sèvres, he continued work on the thermal properties of matter. He designed sensitive thermometers, hygrometers, hypsometers and calorimeters, and measured the specific heats of many substances and the coefficient of thermal expansion of gases. In the course of this work, he discovered that not all gases expand equally when heated and that Boyle's Law is only an approximation, especially at temperatures near a substance's boiling point.

Regnault was also an avid amateur photographer. He introduced the use of pyrogallic acid as a developing agent, and was one of the first photographers to use paper negatives. In 1854, he became the founding president of the Société Française de Photographie.

In 1871, his laboratory at Sèvres was destroyed and his son Alex-Georges-Henri Regnault killed, both as a result of the Franco-Prussian War. He retired from science the next year, never recovering from these losses.

The crater Regnault on the Moon is named after him. Some have suggested that the symbol R for the ideal gas constant is also named after him.