Extracellular vesicle (EV) proteomics holds great promise for non-invasive cancer biomarker discovery. This study presents the first comprehensive proteomic characterization of EVs from tissue, plasma, and urine, focusing on bladder cancer BlCa. EVs were isolated via ultracentrifugation, and their cargo was analyzed, comparing BlCa patients to controls and patients with clear cell renal cell carcinoma (ccRCC) and prostate cancer (PCa), to identify BlCa-specific markers. Urine proved to be a rich source of BlCa EVs, enabling tissue-to-urine molecular tracking and insights into BlCa communication. Differential proteomic analysis revealed distinct BlCa EV profiles compared to healthy donors (HDs), ccRCC, and PCa. Importantly, we identified several candidate biomarkers, including ADAM8 and MMP12, exclusively present in BlCa tissue (50% and 64%, respectively) and urine EVs (83% each). Furthermore, six BlCa urinary EV proteins – ACKR1 (66%) and DHCR7, ERMAP, PTGS2, UBXN4, and GYPC (50% each) – were absent in HDs, ccRCC, and PCa. This study identifies several BlCa-derived EV proteins with strong potential for the development of non-invasive EV-based diagnostics and monitoring strategies for BlCa, potentially contributing to advancing precision medicine approaches for this malignancy .