Objective To explore the pathology and etiology in the patients with cholestatic liver disease.Methods From December 2017 to November 2021, 139 patients with unclarified liver dysfunction manifested as cholestasis were admitted from several hospitals in Anhui province. The clinical data including personal information, laboratory examination and liver biopsy were analyzed. Results Among 139 patients, there were 105 females and 34 males, and the average age was (50.2±11.5) years. All patients underwent liver biopsy, and 121 cases (87.1%) were definitively diagnosed, while the etiology of 18 cases(12.9%) were not clear. One hundred and twenty-one patients were given one or more etiological diagnoses as follows: 85 cases (61.2%) of autoimmune liver disease, 56 cases (40.3%) of primary biliary cholangitis, 15 cases(10.8%) of autoimmune hepatitis, 11 cases(7.9%) of autoimmune hepatitis combined with primary biliary cholangitis, 21 cases (15.1%) of drug-induced liver injury, 5 cases (3.6%) of metabolic fatty liver disease and 2 cases (1.4%) of vanishing bile duct syndrome. In addition, it was found that the alkaline phosphatase(ALP) level in the patients with the autoimmune liver disease was significantly higher [291.0 (225.0, 387.0) U/mL vs. 236.0 (181.0, 293.5) U/mL, P=0.002] than that in other patients.Conclusions Most patients with liver dysfunction and cholestasis were women, and the major diagnosis was autoimmune liver disease, especially primary biliary cholangitis, and the drug-induced liver damage was also one of main causes. ALP could be used to identify whether a patient had autoimmune liver disease.