Extracellular vesicle (EV) surface proteins serve as the fundamental signaling mediators, determining the communication function of EVs. The selective and in-depth exploration of EV surface proteome (surfaceome) is highly compelling but remains technically challenging due to the unique physicochemical properties of surface proteins. These hurdles are further compounded by the inherent heterogeneity of EVs and the low abundance of surface targets in biofluids. Herein, we developed a chemical glycoproteomic strategy, EVscope (EV surface comprehensive one-pot proteomic exploration), for comprehensively profiling the EV surfaceome. The hydrophilic and aromatic lipophilic group-modified magnetic beads enable bias-free EV isolation and in situ oxidation and labeling of cis-diols on surface proteins. Subsequent on-bead processes, including EV lysis, protein digestion, and surface glycopeptide enrichment, collectively maximize surfaceome coverage. Applied to urine samples from 368 individuals with prostate-related diseases, EVscope identified 1,121 EV surface proteins, establishing the largest urine-derived EV surfaceome dataset to date and identifying cathepsin L, integrin alpha 3, fibrinogen beta chain, and tetraspanin-1 as characteristic proteins with potential as noninvasive biomarkers for prostate cancer diagnosis and grading. In summary, this study establishes an innovative methodology for high-resolution EV surfaceome analysis, facilitating noninvasive biomarker discovery and enabling direct detection in population-scale screening.© 2025 Wiley‐VCH GmbH.