Abstract Hepatitis C infection is the main occasional agent of chronic liver disease and might lead to cirrhosis and hepatocellular carcinoma. β-thalassemia and hemodialysis patients are most susceptible to HCV as a result of the continuous blood transfusion. Aim of the study: This study aimed to estimate the quantitative of HCV-RNA in the serum of β- thalassemia patients in Nineveh province using RT-PCR in the GeneXpert system. Methods: We detected anti-HCV using ELISA. Determine the viral RNA using Xpert® HCV viral load technique. Result: The positive detection result of anti-HCV is 48 out of a total of 752 thalassemia patients. Only 12 out of 48 enrolled HCV Ct value with a rate of 1.59% of the total β- thalassemia patients. Conclusions: The prevalence of HCV in thalassemic patients in Nineveh province is less than those found in the other Iraqi provinces or adjacent countries. The routine using of immunobiological and biochemical tests for hepatic enzymes should be the routine pursuit in thalassemia and dialysis clinical centers.