Background: Histological identification of liver nodules between hepatocellular carcinoma (HCC) and other lesions can be full of challenges sometimes, especially in fine-needle aspiration (FNA) biopsies. Our aim was to investigate the efficacy of combined use of Arginase-1, Glypican-3 (GPC-3), heat shock protein 70 (HSP70) and cytokeratin 7 (CK7) in differentiating these lesions in FNA biopsies. Methods: Immunohistochemistry for these four markers was done in 45 HCCs, 22 metastatic carcinoma, 15 intrahepatic cholangiocarcinoma, 12 hepatocellular adenoma, 8 focal nodular hyperplasia, 12 large regenerative nodules arising in cirrhotic livers, and 10 specimens of normal liver tissues. Results: Arginase-1 reactivity was present in 43 of 45 HCCs (96.0%) and all of the benign liver lesions recruited in this study but not in any of MCs and ICCs (P < 0.001). GPC-3 stained 37 of 45 HCCs (82%) and only 1 of 22 MCs (5%), however, all cases of ICCs and the other benign lesions were negative for GPC-3 expression (P < 0.05). HSP70 showed markedly immunoreactivity in progressive tumors, 38 of 45 HCCs (84%), 18 of 22 MCs (82%), and 11 of 15 ICCs (73%), but was negative for all of the benign cases (P < 0.001). All ICCs showed diffused and strong immunostaining for CK7, but only 3 of 45 HCCs (7%) were detected to react with CK7. The combination of the four immunostaining markers for the diagnosis of HCC could raise the sensitivity and specificity to 98% and 100%, respectively. Conclusion: This study demonstrates that Arginase-1 is an extremely effective and specific immunohistochemistry marker for confirming hepaticorigin, while GPC-3 and HSP70 is typically positive in malignant lesions. Our results indicated the accuracy of diagnosis can be enhanced by their combination of these four markers.