Health care workers (HCWs) are at high risk of exposure to hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV transmission due to occupational exposure to injuries, from sharp needles, and scalpels during the execution of their health care duties. We aimed to assess HCV sero-prevalence, and possible risk factors of HCV among HCWs of Kharadar General Hospital, Karachi. Total 300 HCWs were included in the study. Hepatitis C virus antibody was detected through rapid diagnostic test (RDT) then HCV-Ribo-nucleic Acid (RNA) was detected among RDT positive HCWs by using GeneXpert. Structure questionnaire was used for data collection. Data was analyzed by using SPSS 19. Correlation was seen between HCV determinants and positivity by using Pearson chi-square test. A p value of < 0.05 were considered statistically significant. Total 300 HCWs were screened for HCV. Mean age of participants was 30.19 ± 10.55 years and 179 (60%) were female. Both Sero-prevalence and Viraemic prevalence was reported 3 (1%) among study HCWs. Occupational exposure through needle stick injury (NSI) was reported among 172 (57%) HCWs and commonly seen in nursing staff 88 (68.7%), especially at ER 13 (72%). Other significant non-occupational risk factors were ear/nose piercing 175 (58%), dental and surgical procedures 166 (55%). Frequency of HCV among HCWs of study hospital was low, despite high occupational exposure of NSI. Study findings can be used for future prevention of occupational exposure of HCV among HCWs by developing and implementing standard operational procedures and policies.