Objective To investigate the effect of antinuclear antibodies (ANAs) on hormone response in patients with autoimmune hepatitis (AIH)-primary biliary cholangitis (PBC) overlap syndrome (AIH-PBC OS) and AIH-only within half a year. Methods A retrospective analysis of 77 patients with autoimmune liver disease (AILD) admitted to First Clinical Medical College of Lanzhou University from January 2018 to December 2021, all of whom were confirmed by liver biopsy and receiving glucocorticoid treatment. Among them, 46 patients were in AIH-PBC OS group and 31 were in AIH-only group. The general clinical characteristics, liver puncture-related indexes, autoantibodies and immunoglobulin indexes of patients in each group at the time of diagnosis were collected and compared, and the biochemical and immunoglobulin indexes of patients at the time of hormone use and at the time of review within 6 months were also collected, and the hormone response within 6 months was evaluated according to the levels of glutamic transaminase (AST), glutamic alanine transaminase (ALT) and immunoglobulin G (IgG), and the effect of ANAs on hormone response outcomes in both groups over a six-month period was analyzed. Multifactorial ordered logistic analysis was performed to evaluate the effect of ANAs on hormone response between two groups. Results There was no statistically significant difference in the percentage of AIH-PBC OS and AIH-only patients among both ANAs-positive and -negative AILD patients (55.6% vs. 44.4% and 65.6% vs. 34.4%, P>0.05). Among 46 patients with AIH-PBC OS, there were 25 in ANAs-positive group and 21 in ANAs-negative group. The rate of complete hormone response within 6 months was lower than that of ANAs-negative group (44.0% vs. 76.2%), while the rate of hormone non-response was higher than that of ANAs-negative group (20.0% vs. 0), and the difference was statistically significant (P<0.05). There were 20 cases of ANAs-positive and 11 cases of ANAs-negative in the 31 AIH-only patients. There was no statistically significant difference in the results of hormone response within 6 months between the two groups (P>0.05). Multifactorial ordered logistic analysis showed that AIH-PBC OS patients were more likely to have a higher likelihood of 6-month hormone non-response rate in ANAs-positive patients, and the difference was statistically different (P<0.05). And there was no significant effect of ANAs type on hormone response outcome in AIH-only patients (P>0.05). Conclusion AIH-PBC OS ANAs-positive patients have a poor hormone response within half a year. In AIH-only patients, ANAs have no significant effect on hormone response results.