Background: The serological measurement of the anti‐hepatitis C virus antibody is a widely used tool in the first-line diagnosis of HCV infection. Therefore, increasing the testing criteria of these tests is of crucial importance for screening HCV infection. Objectives: The current study aimed to optimize a novel enzyme-linked immuno assay model to detect E2 antigen with or without sample pretreatment in combination with antibodies against core, NS3, NS4, and NS5 antigens of the hepatitis C virus and to compare the performances of these assays with indirect antigen (Ag), biotin/HRP labeled Antigen Sandwich and methods of enzyme-linked immunosorbent assay (ELISA) for their ability to detect HCV. Methods: A total of 107 positive and 415 negative controls from volunteer whole blood donors in Blood Transfusion Organization and 204 blood samples from patients under hemodialysis treatment in Tehran and Bandar Abbas hemodialysis centers are investigated. Six different methods of ELISA test were used to detect anti-HCV antibodies and/or HCV antigens in serum samples. Results: Regarding sensitivity, specificity, and accuracy, E2 Antigen detection alone or combined with antibody detection have the highest accuracy value (99% and 98%, respectively) compared to other methods for antibodies detection. The results of the combined Ag/Ab ELISA test were closer to the results of real-time PCR. Conclusions: This new approach to the detection of antigen and antigen/antibody has better performance criteria concerning the serologic detection of HCV, especially in HD patients who might experience a longer window period.