Background: All blood and blood products should be tested for Hepatitis C virus (HCV) before transfusion to decrease prevalence of HCV. Aim and Objective: The aim of present study is to determine the prevalence of hepatitis c virus (HCV) and to check the accuracy of our laboratory analysis used for HCV testing of blood donors in our blood bank. Material and Methods: The duration of this study is of 2 year since April 2013 to March 2015. All donors are screened during counselling. The blood sample of Total Donor 26,789 including voluntary and replacement were tested for anti HCV antibody by 3 rd generation ELISA (QUALISA).Samples which are reactive or in gray zone were retested by ELISA and also confirmed by rapid HCV card. Results: From total donor 26,789, positive for the HCV were 45 (0.16%). Out of 45, 42 (93.3%) were male, 3(6.6 %,) were female positive. 25 (55.5%) were voluntary and 20 (44.4%) were replacement. Maximum donors come under the age group of 21-30 and 31 -40 and 22 (48.8%) were B RhD positive subjects found. Conclusion: The prevalence of HCV infection is mild (0.16 %) in blood donors in blood bank of PDU medical college and hospital, Rajkot. The prevalence is equal in voluntary and replacement donors. Males are more infective than female.