Objective <p>To evaluate the feasibility and diagnostic value of percutaneous transhepatic cholangioscopy biopsy (PTCB) for identifying the causes of biliary strictures.</p> Methods <p>This retrospective study included 34 patients (18 females and 16 males), with a mean age of 59.4 ± 13&#xa0;years. The study population consisted of patients with suspected malignant biliary strictures on imaging or biliary lesions suspected of malignancy during percutaneous transhepatic endoscopic biliary lithotripsy (PTEBL). The final diagnosis for each patient was confirmed based on surgical pathology results, additional histopathological data, or through close clinical and imaging follow-up for at least one year.</p> Results <p>Among the patients, 20 (58.9%) underwent PTCB alone, while 14 (41.1%) underwent PTCB combined with PTEBL. Biopsy locations included: 12 patients (35.3%) with intrahepatic bile duct, 12 patients (35.3%) with hilar bile duct, and 10 patients (29.4%) with common bile duct. Technical success of PTCB was defined as successful access to the biliary lesion, with the collection of an adequate histopathological tissue sample achieved in 100% of patients. The sensitivity, specificity, and accuracy of PTCB were 90%, 100%, and 97.1%, respectively. Minor complications were observed in 3 patients (8.8%).</p> Conclusion <p>PTCB is a feasible and effective method for diagnosing the causes of biliary strictures, offering high sensitivity, specificity, and accuracy.</p> Graphical abstract