Objective To assess the value of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for diagnosis of extrahepatic biliary lesions. Methods A retrospective analysis was performed on the data of 11 patients with extrahepatic biliary lesions undergoing EUS-FNA in the Third People’s Hospital of Chengdu between January 2012 and December 2016. The result of biopsy was compared with that of surgical finding and follow-up study. Results EUS-FNA was successfully performed on 11 patients with 1 lesions in proximal biliary, 6 in distal biliary, and 4 in ampulla. According to the result of biopsy, 7 lesions were pathologically confirmed as adenocarcinoma, 1 was suspicious of adenocarcinoma, 1 lesion couldn’t be excluded as carcinoma, and 2 were considered to be benign. Compared with the results of surgical pathology and follow-up study, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of EUS-FNA in diagnosis of extrahepatic biliary lesions was 89% (8/9), 100% (2/2), 91% (10/11), 100% (8/8), and 67% (2/3), respectively. One case suffered mild pancreatitis after EUS-FNA without other complications. Conclusion EUS-FNA is a safe, feasible and effective method for diagnosis of lesions in extrahepatic biliary.