Extracellular vesicles (EVs) are shed from all cells and found in accessible biofluids. In addition to abundant tetraspanins (CD9-, CD63-, and CD81), they contain specific markers derived from their source cells. Thus, EVs and their cargo constitute novel biomarkers. However, biofluids contain a mixture of EVs from many different cell types and it is challenging to robustly isolate and characterize specific EV subpopulations. Our new Single Extracellular VEsicle Nanoscopy (SEVEN) assay employs affinity isolation and super-resolution imaging to comprehensively assess EV subpopulations. SEVEN is compatible with both purified EVs and crude biofluids. When we applied SEVEN to crude human plasma, the number of detected tetraspanin-enriched EVs positively correlated with sample dilution in a 50-fold range. We further characterized the size, shape, and molecular tetraspanin content (with corresponding heterogeneities) for CD9-, CD63-, and CD81-enriched EV subpopulations. Additionally, we quantified plasma EVs from four pancreatic ductal adenocarcinoma (PDAC) patients. Since expression of IGF1R is associated with worse outcomes in PDAC patients, we assessed IGF1R-enriched EVs. While only ~0.5% of EVs were IGF1R-enriched, these EVs from PDAC patients were larger, rounder, and contained more tetraspanin molecules, suggestive of a unique pancreatic cancer-enriched EV subpopulation. Altogether, this approach may benefit both general EV research and studies aimed at advancing our biological understanding of human health and disease.