Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that impact human biology by contributing to intercellular communication. To resolve EV heterogeneity and define the EV populations associated with specific biological processes, we developed a method named “EV Fingerprinting” that discerns distinct vesicle populations using dimensional reduction of multi-parametric data collected by quantitative single-EV flow cytometry. After validating this method against synthetic standards, the EV Fingerprinting analysis of highly purified EVs enabled a much more granular resolution of biochemically distinct EV populations than previously established methods. The analysis of EVs produced after molecular perturbation of EV biogenesis through ablation of the GTPase Rab27a and overexpression of the tetraspanin CD63 revealed that EV Fingerprinting reflects the molecular state of a cell. Subsequent analysis of human plasma demonstrates the capacity of EV Fingerprinting to resolve EV populations in complex biological samples and detect tumor-cell derived EVs.