Objective?Clinical immunohistochemistry plays an increasingly important role in pathologic diagnosis. We investigated the usefulness of an immunohistochemical panel of glypican-3 (GPC3), hepatocyte paraffin antigen-1 (HepPar-1), arginase-1 (Arg-1), cytokeratin-19 (CK19), and human epithelial membrane antigen (EMA) for the differential diagnosis of liver tumors. Methods?Two hundred and thirty-five immunohistochemical sections of hepatocellular carcinoma (HCC; 120 cases), intrahepatic cholangiocarcinoma (lCC; 50 cases), combined hepatocellular and cholangiocarcinoma (CHC; 17 cases), metastatic adenocarcinoma (20 cases), and benign liver lesions (28 cases) were obtained from the Department of Pathology at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The sensitivity and specificity of the combined biomarkers GPC3/HepPar-1/Arg-1/CK19/EMA for the differential diagnosis of HCC, lCC, and CHC were calculated and analyzed retrospectively. Results?The combined biomarkers GPC3+/CK19– had the highest specificity (98.3%) for diagnosing HCC, with a sensitivity of 60.0%. The specificity of GPC3–/HepPar-1–/Arg-1–/CK19+/EMA+ for diagnosing lCC was 93.0%, with a sensitivity of 76.0%. The specificity of GPC3+/HepPar-1+/Arg-1+/CK19+/EMA+ for diagnosing CHC was 95.9%, with a sensitivity of 52.9%. Conclusion?The combined biomarkers GPC3/HepPar-1/Arg-1/CK19/EMA greatly improved the specificity of liver tumor diagnosis. We believe that clinical pathological work could improve the original determination of liver nodules.