Detection of extracellular vesicles (EVs) cargoes from biofluids has drawn more attention in the field of translational and clinical medicine. EV proteomics is particularly a promising tool in discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not well compatible with clinical settings due to inefficient EV isolation methods and tedious sample preparation processes. To further implement highly efficient EV proteome analysis, this study established a one-pot analytical pipeline based on a robust EV isolation approach, EV total recovery and purification (EVtrap), to detect urinary EV proteome in a rapid and sensitive manner. By incorporating solvent-driven protein capture and on-bead digestion, the one-pot EV proteomics pipeline avoided any sample transfer step and largely reduced the complexity of the peptide preparation process for bottom-up proteomic analysis. Moreover, a shorter digestion time was practicable in this novel pipeline, which enables a whole EV proteome analysis to be completed within one day. In comparison with the more time-consuming conventional workflow, the one-pot pipeline was able to obtain a higher peptide yield and identify similar numbers of unique EV proteins in 1 mL of urine. Finally, we applied the one-pot pipeline to monitor potential biomarkers in urinary EVs of bladder cancer patients. A total of 2,774 proteins and ~20,000 peptides were identified in 53 urine samples using a 15-min gradient direct data-independent acquisition (directDIA). Several known bladder cancer-associated protein markers were successfully identified and showed significantly higher abundance in patient-derived EVs. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.