Born the son of a politician at Haarlem in the Netherlands, de Vries studied botany at Leiden and Heidelberg. He became an expert on the Netherlands flora and later turned his attention from classification to physiology and evolution. He entered Julius von Sachs's laboratory at Würzburg University, where he conducted important experiments on the water of plant cells. He demonstrated that the pressure (turgor) of the cell fluid is responsible for about 10% of extension growth, and introduced the term plasmolysis to describe the condition in nonturgid cells in which the cell contents contract away from the cell wall. His work in this field led to Jacobus van't Hoff's theory of osmosis.

During the 1880s, de Vries became interested in heredity. In 1889 he published Intracellular Pangenesis, in which he critically reviewed previous research on inheritance and advanced the theory that elements in the nucleus, ‘pangenes’, determine hereditary traits. To investigate his theories, he began breeding plants in 1892 and by 1896 had obtained clear evidence for the segregation of characters in the offspring of crosses in 3:1 ratios. He delayed publishing these results, proposing to include them in a larger book, but in 1900 he came across the work of Gregor Mendel, published 34 years earlier, and announced his own findings. This stimulated both Karl Correns and Erich von Tschermak-Seysenegg to publish their essentially similar observations.

De Vries' work on the evening primrose, Oenothera lamarckiana, began in 1886 when he noticed distinctly differing types within a colony of the plants. He considered these to be mutants and formulated the idea of evolution proceeding by distinct changes such as those he observed, believing also that new species could arise through a single drastic mutation. He published his observations in The Mutation Theory (1901–03). It was later shown that his Oenothera ‘mutants’ were in fact triploids or tetraploids (i.e. they had extra sets of chromosomes) and thus gave a misleading impression of the apparent rate and magnitude of mutations. However, the theory is still important for demonstrating how variation, essential for evolution, can occur in a species.

De Vries was professor of botany at Amsterdam from 1878 to 1918 and was elected a fellow of the Royal Society in 1905.