Abstract Background Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are severe diseases with heterogenous manifestations, which affect people of all populations and age groups. Diagnosis and differentiation from other liver diseases such as primary sclerosing cholangitis (PSC) remain a substantial challenge for hepatologists. Since AIH and PBC are caused by different sets of autoantibodies, we previously developed a line blot for the parallel detection of all relevant parameters. We now added the AIH-specific antigen filamentous actin (F-actin) to this established liver immunoassay profile. Methods Sera from 15 patients with AIH, 15 patients with overlap AIH/PBC or AIH/PSC, 32 patients with PSC, 53 patients with PBC, 4 patients with non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), 30 patients with hepatitis B infection and 79 patients with hepatitis C infection as well as sera from 150 blood donors were examined using the EUROLINE line immunoassay system (EUROIMMUN autoimmune liver diseases 9 Ag plus F-actin (IgG); contains the PBC-relevant antigens AMA-M2, M2-3E (BPO), Sp100, PML and gp210, the AIH-associated antigens LKM-1, LC-1 and SLA/LP and F-actin as well as Ro52). The newly included F-actin antigen was polymerized from human globular actin (G-actin) and assessed by sedimentation analysis and dynamic light scattering. The scan software EUROLineScan (EUROIMMUN) was used to automatically determine presence and intensity of the resulting bands on the blot. Results Quality assessment revealed successful polymerization of F-actin. Autoantibodies against this newly developed F-actin antigen were detected in 53.3% of sera from AIH patients and 40% of AIH+PBC/AIH+PSC overlap patients. 93% of these positive sera did not contain other autoantibodies associated with AIH. Only one PSC patient (3.1%), two patients with hepatitis C (2.5%) and two blood donors (1.3%) also showed anti-F-actin positivity. All tested sera of PBC, NAFLD/NASH and hepatitis B patients were evaluated as anti-F-actin negative. Conclusions The addition of F-actin to the established multiparametric EUROLINE profile for the simultaneous and monospecific detection of AIH- and PBC-relevant autoantibodies is a great advance for routine diagnostics of patients with symptoms associated with various liver diseases.