Extracellular vesicles (EVs) are released from all cells. Bile directly contacts bile duct tumor; bile-derived EVs may contain high concentrations of cancer biomarkers. We performed a proteomic analysis of human bile-derived EVs and identified a novel biomarker of cholangiocarcinoma (CCA). EVs were isolated using ultracentrifugation, and chelating agents, ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (EDEG) and phosphate buffered saline (PBS) were used as dissolution solutions. Bile was collected from 10 CCA and 10 choledocholithiasis (stones) cases. Proteomic analysis was performed; subsequently, ELISA was performed using the candidate biomarkers in a verification cohort. The vesicles isolated from bile had a typical size and morphology. The expression of exosome markers was observed. RNA was more abundant in the EDEG group. The proportion of microRNA was higher in the EDEG group. EDEG use resulted in the removal of more contaminants. Proteomic analysis identified 166 proteins as CCA-specific. ELISA for Claudin-3 revealed statistically significant difference. The diagnostic accuracy was AUC 0.945 and sensitivity and specificity were 87.5%. We report the first use of EDEG in the isolation of EVs from human bile and the proteomic analysis of human bile-derived EV-proteins in CCA. Claudin-3 in bile-derived EVs is a useful biomarker for CCA.