Objective: To investigate the diagnostic value of SpyGlass choledochoscopy system in indeterminate stricture of hilar bile duct. Methods: A retrospective analysis was conducted on 146 patients with indeterminate stricture of the hilar bile duct who underwent endoscopic retrograde cholangio-pancreatography (ERCP) and SpyGlass examinations at our hospital between January 2018 and December 2022. The study recorded the visual impressions of the lesions, direct visualization-guided biopsy pathology, and conventional brush cytology results obtained using SpyGlass system. The sensiti-vity, specificity, and accuracy of different diagnostic methods were compared, taking pathological diagnosis as the gold standard and considering long-term follow-up results as well. Results: Out of the 146 patients, the procedure was successful in 145 cases (99.3%). The average procedure time was （51.5±16.9） min. The sensitivity, specificity, and accuracy of SpyGlass visual impression diagnosis for indeterminate biliary stricture were 83%, 84%, and 83%, respectively, outperforming brush cytology results (47%, 100%, and 52%). The combination of visual diagnosis and SpyBite biopsy yielded sensiti-vity of 91%, specificity of 63%, and accuracy of 85%. Adverse events occurred in 24.7% of cases, all of which were clinically mild. Conclusions: SpyGlass choledochoscopy has a high accuracy and safety in differential diagnosis of indeterminate stricture of the hilar bile duct. Its widespread application is warranted.