Background and Aims: The burden of HIV infection and disease continues to be a major global public health concern all over the world. This study was conducted to measure IgG antibody against EBV viral capsid antigen (EBV-VCA IgG), Herpes Simplex Type1, and 2 to determine the seroprevalence of this infection in HIV-positive population. Materials and Methods: A Cross-sectional study between March and August 2016 was conducted in keyvan virology laboratory enrolling 84 HIV-positive patients with different age and sexes. Enzyme-linked immunosorbent assay (ELISA) was used for determination of IgG antibody against Herpes Simplex Type1, Type 2 viruses, EBV and HIV viral load detected in plasma by Real time-PCR in obtained samples from HIV-positive patients. Results: The overall seroprevalence was 66.7% for anti-HSV-1 IgG, 14.3% for anti-HSV-2 IgG, and 94% for anti-EBV IgG. There was no significant difference between the sex groups for HSV-1 and -2, EBV and HIV load and their IgG level. This study also showed a correlation between the age, and the antibody titers only for HSV-1 and -2 with P=0.030 and P= 0.024, respectively. Conclusions: In our study, the seroprevalence of EBV and HSV-1 IgG were higher in HIVpositive patients. It can be derived that HSV-2 virus is not major coinfection in Iran, thus requires less attention than others, but annually monitoring needs for proper health care programs.