A new immunoblot assay, composed of five Epstein-Barr virus (EBV)-encoded recombinantproteins virus capsid antigen [VCA] gp125, p19, p22, early antigen [EA], and EBNA-1 IgG, wasused to manifest the EBV infection and look at the antibody pattern to EBV proteins in the serumof both autoimmune disorders and Thalassemia patients and compare the observations with thosein normal healthy controls. Serum samples from 35 rheumatoid arthritis patients, 20 SLE, 20autoimmune hypothyroid diseases, 35 Thalassemia patients and 20 healthy controls were testedfor EBV IgG antibodies by an immunoblot assay (Euroline). The results showed that the highpercentage recorded was 50% in acute infection. Followed by 30% at late infection, while latephase with loss EBNA-1 and reactivated infection were 10% compared to the normal healthycontrols. Our study showed an increased EBV activation among the autoimmune patient groupscompared to the normal healthy controls. Further studies are required to delineate the associationbetween the etiology of autoimmune disorders and EBV.