Trypsin is an ancient protease best known as a digestive enzyme in animals, and traditionally believed to be absent in plants and protists. Here, we surveyed the distribution, diversity, evolution and potential functions of trypsin genes in global ocean phytoplankton, the major primary producers in the aquatic ecosystem. Our analysis indicates that trypsin genes are widely distributed both taxonomically and geographically in marine phytoplankton. Furthermore, by systematic comparative analyses we documented lineage-specific diversity and expansion of trypsin genes in the evolution of marine phytoplankton. Genome-wide analyses revealed that trypsin genes were more prevalent in diatoms than in other lineages. Moreover, the expression of trypsin genes in diatom tended to be more responsive to environmental stimuli. The duplication and neofunctionalization of trypsin genes may be important in diatoms to adapt to dynamical environmental conditions, contributing to diatoms’ dominance in the coastal oceans. This work advances our knowledge on the distributions and neofunctionalizations of this ancient enzyme and creates a new research direction in the phytoplankton biology.