A large number of healthy donors being falsely labeled as carriers of HCV (false-positive) due to the cross reaction. The low specificity of the enzyme immunoassay (EIA) tests for the anti-HCV antibodies is responsible for this event. To examine the cross reactivity values of different Hepatitis C Virus antigens and purpose a solution to detect true positive results by third generation of ELISA. A total of 988 blood samples were participated in this study and two kinds of ELISA tests were used in all of the samples: single-antigen ELISA and third generation of ELISA. Tests set up procedures were perfomed by appropriate concentrations of antigens and conjugated titer for each test. Thereafter, all true positive samples were examined to determine IgG subtypes. The highest antibody response was against core HCV protein (85%) followed by NS4, NS5 and NS3 respectively. It was observed that high percentage (95%) of samples with S/Co > 4 in third generation of ELISA had positive RIBA tests (p value < 0.001). It was also demonstrated that NS3 and NS4 antigens were diagnosed simultaneously more (in compare with NS5 and Core24) in S/Co > 4 samples (p value = 0.006). The highest produced antibody during HCV infection was IgG1, significantly (P=0.023). In conclusion, if the S/Co of third generation of ELISA is more than four or the samples were positive in NS3 and NS4 single antigen ELISA tests, the RIBA test will be positive which indicated true positive results.