De Duve was born of an estate agent Alphonse de Duve and wife Madeleine Pungs in the village of Thames Ditton, near London. His parents fled Belgium at the outbreak of the First World War. After the war in 1920, at age three, he and his family returned to Belgium. He was a precocious boy, always the best student (primus perpetuus as he recalled) in school, except for one year when he was pronounced "out of competition" to give chance to other students.

Its true biological importance was not known and the name itself was ignored. At the time de Duve joined Bouckaert at Leuven university to work on insulin, it still remained a mystery. Insulin was the first commercial hormonal drug originally produced by the Eli Lilly and Company since 1921, but their extraction suffered from impurity which caused mild hyperglycaemia, the very opposite of what they expected. In May 1944 de Duve realised that the insulin impurity could be removed by crystallisation. He demonstrated that the insulin produced of ELi Lilly was contaminated by the impurity, whereas that of the Danish Novo was not. The Eli Lilly insulin caused initial hyperglycaemia, and the Novo insulin did not when he injected them into rats. His experiments were published in 1947, following which the Eli Lilly upgraded its method to produce purified insulin. By then he had joined Carl and Gerti Cori's at Washington University in St. Louis, where he worked with a fellow researcher Earl Wilbur Sutherland, Jr. (who later won the Nobel Prize in Physiology or Medicine in 1971).

The hormone glucagon was discovered by C.P. Kimball and John R. Murlin in 1923 as a hyperglycaemic (blood-sugar elevating) substance along the pancreatic extracts.

He was educated by the Jesuits at Onze-Lieve-Vrouwinstituut in Antwerp, before studying at the Catholic University of Leuven in 1934. He wanted to specialize in endocrinology and joined the laboratory of the Belgian Physiologist Joseph P. Bouckaert.

During his last year at medical school in 1940, the Germans invaded Belgium. He was drafted to the Belgian army, and posted in southern France as medical officer. There, he was almost immediately taken as prisoner of war by Germans. His ability to speak fluent German and Flemish helped him outwit his captors. He escaped back to Belgium in an adventure he later described as "more comical than heroic".

He immediately continued his medical course, and obtained his MD in 1941 from Leuven. After graduation, de Duve's primary research was on insulin and its role in glucose metabolism. He made an initial discovery that a commercial preparation of insulin was contaminated with another pancreatic hormone, the insulin antagonist glucagon.

De Duve married Janine Herman on 30 September 1943. Together they had had two sons, Thierry and Alain, and two daughters, Anne and Françoise. Janine died in 2008, aged 86.

However, laboratory supplies at Leuven were in shortage, therefore he enrolled in a programme to earn a degree in chemistry at the Cancer Institute. His research on insulin was summed up in a 400-page book titled Glucose, Insuline et Diabète (Glucose, Insulin and Diabetes) published in 1945, simultaneously in Brussels and Paris. The book was condensed into a technical dissertation which earned him the most advanced degree at the university level agrégation de l'enseignement supérieur (an equivalent of a doctorate – he called it "a sort of glorified Ph.D.") in 1945. His thesis was followed by a number of scientific publications.

He subsequently obtained a MSc in chemistry in 1946, for which he worked on the purification of penicillin.

In March 1947 de Duve joined the faculty of the medical school of the Catholic University of Leuven teaching physiological chemistry. In 1951 he became full professor. In 1960, Detlev Bronk, the then President of the Rockfeller Institute (what is now Rockefeller University) of New York City, met him at Brussels and offered him professorship and a laboratory. The rector of Leuven, afraid of entirely losing de Duve, made a compromise over dinner that de Duve would still be under part-time appointment with a relief from teaching and conducting examinations. The rector and Bronk made an agreement which would initially last for five years. The official implementation was in 1962, and de Duve simultaneously headed the research laboratories at Leuven and at Rockefeller University, dividing his time between New York and Leuven.

De Duve was one of the founding members of the Belgian Society of Biochemistry and Molecular Biology, established on 15 September 1951.

De Duve was the first to hypothesise that production of insulin to decrease blood sugar level stimulated the uptake of glucose in the liver, and that there is a balanced production of insulin and glucagon to maintain normal blood sugar level. His idea was ridiculed at the time. But his rediscovery of glucagon confirmed his ideas. In 1953 he experimentally demonstrated that glucagon did influence the production and thus uptake of glucose.

He was skeptical of referring to them as microbodies because, as he noted, "too little is known of their enzyme complement and of their role in the physiology of the liver cells to substantiate a proposal at the present time". He suggested that these enzymes belonged to the same cell organelle, but different from previously known organelles. But it would take some years before he publicised his hypothesis, as strong evidences were still lacking. In 1955 his team demonstrated similar cell fractions with same biochemical properties from the ciliated protozoan Tetrahymena pyriformis, from which it was indicated that these particles were new cell organelles unrelated to mitochondria. He presented his discovery at a meeting of the American Society for Cell Biology in 1955, and formally published in 1966 in which he created the name peroxisomes for the organelles as they are involved in peroxidase reactions. In 1968 he achieved the first large-scale preparation of peroxisomes, confirming that l-α hydroxyacid oxidase, d-amino acid oxidase, and catalase were all the unique enzymes of peroxisomes.

De Duve won the Francqui Prize for Biological and Medical Sciences in 1960, and the Nobel Prize for Physiology or Medicine in 1974. King Baudouin of Belgium honoured him to Viscount in 1989. He was the recipient of the Canada Gairdner International Award in 1967, and the Dr H.P. Heineken Prize for Biochemistry and Biophysics in 1973 from the Royal Netherlands Academy of Arts and Sciences.

De Duve is remembered as an Inventor of important scientific terminology. He coined the word lysosome in 1955, peroxisome in 1966, and autophagy, endocytosis, and exocytosis in one instance at the Ciba Foundation Symposium on Lysosomes held in London during 12–14 February 1963, while he, "was in a word-coining mood."

In 1969, the Leuven university was split into two separate universities. He joined the French-speaking side of Université catholique de Louvain. He took emeritus status at Université catholique de Louvain in 1985 and at Rockefeller in 1988, though he continued to conduct research. Among other subjects, he studied the distribution of enzymes in rat liver cells using rate-zonal centrifugation. His work on cell fractionation provided an insight into the function of cell structures. He specialized in subcellular biochemistry and cell biology and discovered new cell organelles.

De Duve founded a multidisciplinary biomedical research institute at Université catholique de Louvain in 1974, originally named the International Institute of Cellular and Molecular Pathology (ICP)

He was elected a foreign associate of the US National Academy of Sciences in 1975. He won the Harden Medal of the Biochemical Society of Great Britain in 1978; the Theobald Smith Award from the Albany Medical College in 1981; the Jimenez Diaz Award in 1985; the Innovators of Biochemistry Award from Medical College of Virginia in 1986; and the E. B. Wilson Medal from the American Society for Cell Biology in 1989.

He was also a member of the Royal Academies of Medicine and the Royal Academy of Sciences, Arts, and of Literature of Belgium; the Pontifical Academy of Sciences of the Vatican; the American Academy of Arts and Sciences; the French National Academy of Medicine; the Academy of Sciences of Paris; the Deutsche Akademie der Naturforscher Leopoldina; the American Philosophical Society. He was elected a Foreign Member of the Royal Society (ForMemRS) in 1988. In addition, he received honorary doctorates from eighteen universities around the world.

De Duve was granted the rank of Viscount in 1989 by King Baudouin of Belgium. He was also a recipient of Francqui Prize, Gairdner Foundation International Award, Heineken Prize, and E. B. Wilson Medal. In 1974 he founded the International Institute of Cellular and Molecular Pathology in Brussels, eventually renamed the de Duve Institute in 2005. He was the founding President of the L'Oréal-UNESCO Awards for Women in Science.

He remained its President until 1991. On his 80th birthday in 1997 it was renamed the Christian de Duve Institute of Cellular Pathology. In 2005 its name was further contracted to simply the de Duve Institute.

He strongly supported biological evolution as a fact, and dismissive of creation science and intelligent design, as explicitly stated in his last book, Genetics of Original Sin: The Impact of Natural Selection on the Future of Humanity. He was among the seventy-eight Nobel laureates in science to endorse the effort to repeal the Louisiana Science Education Act of 2008.

De Duve's life, including his work resulting in a Nobel Prize, and his passion for biology is the subject of a documentary film Portrait of a Nobel Prize: Christian de Duve (Portrait de Nobel : Christian de Duve), directed by Aurélie Wijnants. It was first aired on Eurochannel in 2012.

De Duve died on 4 May 2013, at his home in Nethen, Belgium, aged 95. He decided to end his life by legal euthanasia, performed by two doctors before his four children. He had been long suffering from cancer and atrial fibrillation, and his health problems were exacerbated by a recent fall in his home. He is survived by two sons and two daughters; two brothers, Pierre and Daniel; seven grandchildren; and two greatgrandchildren.