The etiological diagnosis of biliary stricture remains a clinical challenge. Currently, endoscopic retrograde cholangiopancreatography (ERCP)-guided biliary biopsy is the most commonly used technique. This retrospective study aimed to evaluate the clinical value of percutaneous transhepatic intraluminal forceps biopsy (TIFB) in patients with biliary stricture after ERCP failure. The clinical data of 240 consecutive patients with biliary strictures who sought further etiologic diagnosis at our center between April 2014 and January 2020 were collected. After the exclusion of 197 patients who underwent ERCP-guided biopsy, 43 patients who received TIFB after ERCP failure were included in the study. The primary outcomes were technical success, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. Secondary outcomes included procedure duration, radiation exposure, liver function [total bilirubin (TB), direct bilirubin (DB), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] preoperatively and at 2 weeks postoperatively, and complications. The technical success rate of TIFB was 100%. The diagnostic sensitivity, specificity, accuracy, PPV, and NPV of TIFB were 82.35%, 100%, 86.05%, 100%, and 60%, respectively. The accuracy of TIFB was significantly higher for cases with suspicious biliary tract invasion on imaging and intrabiliary malignant origin than it did for cases with no suspicious biliary tract invasion on imaging or extrabiliary malignant origin (P=0.007 and P=0.003, respectively). Only intrabiliary malignant origin (P=0.02) was an independent contributing factor for a true positive result in TIFB diagnosis. The mean procedure duration was 19.3 minutes and the mean radiation exposure was 315.6 mGy. All liver function markers were significantly reduced after 2 weeks (all P<0.001). Three (6.97%) complications occurred, including 1 (2.33%) case of cholangitis and 2 (4.65%) cases of hemobilia. Percutaneous TIFB is an effective method with high sensitivity and accuracy for the etiological diagnosis of biliary stricture after ERCP failure.