Extracellular vesicles (EVs) are promising non-invasive biomarkers, but their clinical application is limited by inefficient isolation and downstream analysis. In this study, we developed a biomimetic magnetic cilia channel chip (MagCilia-chip) for the efficient capture and proteomic profiling of urinary EVs. Tim4-functionalized magnetic microcilia specifically captured phosphatidylserine-positive EVs, while magnetic-field-driven three-dimensional rotation enhanced EV–microstructure interactions and enabled on-chip EV lysis and enzymatic digestion. Data-independent acquisition mass spectrometry identified 2,903 EV-derived proteins from only 50 μL of urine. Comparative proteomic profiling was performed using an independent discovery cohort comprising 12 patients with prostate cancer and 8 post-treatment patients, resulting in the identification of 368 differentially abundant EV proteins. An independent parallel reaction monitoring cohort comprising 19 prostate cancer and 9 post-treatment samples was used for targeted validation. Differential abundance of C9, COL6A3, and VTN was confirmed, and the resulting three-protein candidate panel was evaluated using multiple machine-learning classifiers. This dataset supports the development of urinary EV-based proteomic approaches for prostate cancer research and post-treatment monitoring.