Abstract: Objective To explore the clinical significance of multiple autoantibodies in the diagnosis and treatment of primary biliary cholangitis （PBC）. Methods A total of 58 patients with PBC, 60 patients with viral hepatitis, and 60 healthy people were recruited and assigned into three groups. The antinuclear antibody （ANA） and anti-mitochondrial antibody （AMA） were detected by indirect immunofluorescence assay （IIF）. The anti-mitochondrial antibody M2 type （AMA-M2）, anti-gp210 and anti-sp100 were determined by the linear immunoblotting （LIA）. The significance of single and combined detection of different autoantibodies in diagnosis and treatment of PBC was evaluated. Results The positive rate of ANA significantly differed among the PBC, viral hepatitis and normal control groups （all P < 0.05）. The positive rate of ANA in the PBC group was the highest and that in the normal control group was the lowest. The positive rate of ANA significantly differed between any two groups （all P < 0.017）. For individuals with positive ANA, the proportion of patients with medium and high titer （1∶320 or above） in the PBC group was significantly higher compared with those in the viral hepatitis and normal control groups, respectively （both P < 0.017）, whereas the proportion of low titer （1∶100） was significantly lower than that in the viral hepatitis group （P < 0.017）. Among 54 patients with positive ANA in the PBC group, the ANA fluorescence karyotype mainly consisted of cytoplasmic and granule type （43%） and centromere type （20%）. The positive rates of ANA, AMA, AMA-M2, anti-gp210 and anti-sp100 and combined detection of ANA, AMA, AMA-M2, anti-gp210 and/or anti-sp100 in the PBC group were significantly higher compared with those in the normal control and viral hepatitis groups （all P < 0.017）. In the PBC group, the positive rate of combined detection of anti-gp210 and anti-sp100 was significantly higher than single detection of anti-gp210 and anti-sp100 （both P < 0.05）. The sensitivity of ANA （1∶320 or above）, AMA, AMA-M2, anti-gp210 and anti-sp100 and combined detection in the PBC patients was 88%, 85%, 78%, 29%, 16% and 90%, 92%, 97%, 98%, 100% and 92% for the specificity, 91%, 96%, 98%, 94%, 9/9 and 91% for the positive predictive value, and 89%, 87%, 82%, 59%, 55% and 90% for the negative predictive value, respectively. Conclusions In the clinical screening of PBC, detection of liver autoantibody spectrum should be supplemented for patients presenting with medium and high titer of cytoplasmic granule type after receiving ANA detection alone. Combined detection of multiple autoantibodies can improve the detection rate of PBC, providing more diagnostic basis for clinical practice.