De Duve was born at Thames Ditton in southern England and educated at the Catholic University of Louvain where he obtained his MD in 1941. After holding brief appointments at the Nobel Institute in Stockholm and at Washington University he returned to Louvain in 1947 and was appointed professor of biochemistry in 1951. From 1962 to 1988 he held a similar appointment at Rockefeller University in New York.

In 1949 de Duve was working on the metabolism of carbohydrates in the liver of the rat. By using centrifugal fractionation techniques to separate the contents of the cell, he was able to show that the enzyme glucose-6-phosphatase is associated with the microsomes – organelles whose role was only speculative until de Duve began this work. He also noted that the process of homogenization led to the release of the enzyme acid phosphatase, the amount of which seemed to vary with the degree of damage inflicted on the cells. This suggested to de Duve that the enzyme in the cell was normally enclosed by some kind of membrane. If true, the supposition would remove a problem that had long troubled cytologists – namely how it was that such powerful enzymes did not attack the normal molecules of the cell. This question could now be answered by proposing a self-contained organelle, which neatly isolated the digestive enzymes. Confirmation for this view came in 1955 with the identification of such a body with the aid of the electron microscope. As its role is digestive or lytic, de Duve proposed the name ‘lysosome’. The peroxisomes (organelles containing hydrogen peroxide in which oxidation reactions take place) were also discovered in de Duve's laboratory.

For such discoveries de Duve shared the 1974 Nobel Prize for physiology or medicine with Albert Claude and George Palade.