Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.