Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.