Background: The detection of hepatitis C virus (HCV) RNA is essential to diagnose acute and chronic hepatitis C infection, therapy administration and follow up of treatment, and for the HCV genotype characterization. Materials and Methods: In current study fifty HCV infected patients were included; blood samples were collected from each and RT-nested PCR for HCV RNA detection were done. Results: out of 50 patients 29 were males (58.0 %) and 21 were females (42.0%). Their age range was from 5 to 67 years, and they were distributed into four groups 1. 1-20 years, 2. 21-40 years, 3. 41-60 years, and 4. >61 years), three of them were in group 1 (6.0%), 30 in group two (60.0%), 14 in group three (28.0%), and 3 in group four (6.0%). All samples were subjected to Qualitative HCV RT-nested PCR, and all of it were positive (100.0%). Conclusion: Molecular diagnosis of HCV is important for HCV treatment and genotype detection, in current study HCV RT-nested PCR was successfully applied for HCV detection in Libya.