AbstractExtracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub‐100 nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Now, single‐EV flow cytometry analysis is introduced to realize single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by target‐initiated engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, statistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines, and the cancer cell‐derived EVs among the heterogeneous EV populations are successfully recognized. Thus, our approach holds great potential for various biological and biomedical applications.