Background: Hepatocellular carcinoma (HCC) is considered the sixth most common cancer in the world. Epithelial membrane antigen (EMA) is a glycoprotein which is associated with the tumor formation and has an important role in metastasis. During the process of tumor cells invasion and transformation, the changes in the glycolization of EMA affect on the tumor biological behavior. The aim of the present study is the identification of EMA in serum samples in patients with HCC, detection of EMA in serum samples in patients with HCC and determination of detection rate of EMA in serum samples in patients with HCC. Methods: Total number of 100 patients with HCC and 35 healthy controls were recruited. Clinical and laboratory data were measured in patients and controls. Serum EMA rate was assessed using western blotting and ELISA. Results: HCC group had higher liver enzymes, bilirubin and INR and a lower to mean platelet counts and albumin levels than controls. HCC group was significantly higher (P < 0.001) in AFP level than that of Control group. Also, there was a highly significant difference (P < 0.001) between the level of EMA in HCC group and control group. Epithelial membrane antigen was identified in serum samples of hepatocellular carcinoma by using monoclonal antibody (EMA mAb) and western blot technique at 130 kDa. Conclusions: Since, the EMA was detected by ELISA with a sensitivity of 81% and a specificity of 100%, EMA can be used in HCC diagnosis.