Background: Chronic infection with hepatitis C virus is one of major risk factors in the development of the hepatocellular carcinoma. Cancer stem cells (CSCs) particularly with biomarkers (CD133, CD90 and CK19) show higher ability for self-renewal, differentiation and also tumorigenesis. Objectives: to evaluate the role of CD133, CD90 and CK19 in detection and prognosis of HCC on top of HCV infection. Methodology: This study enrolled 75 participants; 30 HCC patients secondary to HCV infection, 15 HCV patients before receiving the antiviral drug treatment, 15 HCV patients who completed the course of antiviral therapy for 3 months and 15 age and sex-matched apparently healthy volunteers. HCV was detected and quantitated by Quantitative Real-Time PCR and the expression level of CD133, CD90 and CK19 on PBMC was determined by Quantitative Real-Time reverse transcription PCR. Results: this study showed high significant increase in mean expression values of CD133, CD90 & CK19 was in patients with HCC than other studied groups. Also, they were highly significantly increased in HCV patients before than those after receiving treatment and their expression showed high significant positive correlation with HCV load. Conclusion: There were higher expressions of CSC biomarkers (CD133, CD90 and Ck19) in patients with HCC in comparison with those with hepatitis C infection. Their expressions had good diagnostic and prognostic values for HCC.