HEV infection is predominantly spread via the fecal-oral route; however, due to the presence of HEV RNA in the serum of healthy blood donors, there is a possibility of the transmissibility of HEV infection through blood. Multi-transfused thalassemia patients are one of the high risk groups for blood borne viruses. In this study, we evaluated the prevalence of HEV antibodies and HEV-RNA in thalassemia patients with HCV infection. 120 anti-HCV positive thalassemia patient serum samples from Tehran province during April-June 2019 were assessed for the presence of total anti-HEV antibodies using of HEV Ab ELISA kit. All serum samples were assayed by Nested RT-PCR to detect HEV-RNA. The results of ELISA test showed that 2 out of 120 (1.67%) samples were positive for anti-HEV Ab. There was no statistically significant difference between anti-HEV antibody prevalence rate and sex, age and other risk factors. None of 120 (0.00%) samples were positive for HEV-RNA by Nested RT-PCR. Seroprevalence of HEV in our study group was 1.67% which is less than HEV seroprevalence rate in Iranian general population. Therefore, it can be conclude that transmission of HEV infection via blood transfusion seems to be uncommon in Iran and the fecal-oral route can be the predominant mode of transmission in Iran; however, more studies are required to confirm this issue.