Objective To evaluate endoscopic ultrasonography (EUS) in the diagnosis and preoperative assessment of hilar cholangiocarcinoma. Methods Data of consecutive patients with hilar biliary strictures who underwent EUS and were confirmed by postoperative pathology from April 2016 to December 2019 were collected in the retrospective study. The clinical information, EUS image characteristics and lymph nodes of patients were recorded and analyzed. Results A total of 58 patients were finally included in our study. Hilar cholangiocarcinoma of EUS image was characterized by heterogeneous hypoechoic, non-rich blood supply masses and (or) asymmetric thickening of bile duct wall. Among the 58 cases, 45 cases (77.6%) were manifested as masses and 32 cases (55.2%) were presented as thickening of bile duct wall. Nineteen cases (32.8%) had both manifestations above. There were 10 cases of vascular invasion detected by EUS scanning, including 3 cases of portal vein invasion, 4 cases of hepatic artery invasion, 3 cases of invasion of both loci. Postoperative pathology confirmed 14 cases of vascular invasion, with the diagnostic coincidence rate of 71.4% (10/14). A total of 101 lymph nodes were found in 53 patients by EUS scanning. The malignant lymph nodes presented hypoechoic, round or oval shape, and homogeneous echo. Compared with benign lymph nodes, malignant lymph nodes had higher morphological score (11.41±0.6 VS 9.01 ± 0.15, P<0.001), but there was no significant difference in size (13.29±0.90 mm VS 11.87±0.56 mm, P=0.28). According to the malignancy criteria of EUS lymph nodes (morphological score≥12), the accuracy, the sensitivity, the specificity, the positive predictive value and the negative predictive value of EUS for malignant lymph nodes were 92.1%(93/101), 76.5% (13/17), 95.2% (80/84), 76.5% (13/17) and 95.2% (80/84) , respectively. Conclusion EUS can show the whole extrahepatic bile duct and part of intrahepatic bile duct, which is helpful to determine the location of tumor in the diagnosis of hilar cholangiocarcinoma. Moreover, EUS is helpful to diagnose hilar cholangiocarcinoma, which is of guiding significance in operative decisions.