Chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) resulted in dysfunction of the immune response, in particular, dendritic cells (DCs). Stimulation and reactivation of DCs could restore the immune responses in this disease. Our previous study revealed that a synthetic double-stranded RNA (polyI:C) recognized by toll-like receptor 3 (TLR3) induced a potent innate immune response and had an impact on DCs both ex vivo and in vivo. The current study was aimed to generate and maturate DCs from peripheral blood mononuclear cells (PBMCs) of healthy controls and patients with HCV and HCC. Fresh peripheral blood (PB) was collected from healthy controls, patients with chronic HCV or patients with hepatocellular carcinoma (HCC). PBMCs were isolated and cultured for 6 days in RPMI-1640 supplemented with GM-CSF and rhIL-4 (10 ng/mL each). Poly(I:C) was added to the culture on day 6 and the cells were harvested on day 7. Phenotypic analysis of DCs and their maturation markers were assessed using flow cytometry. DCs were generated from adherent PBMCs of healthy donors or patients with HCV or HCC. Interestingly, the addition of poly(I:C) induced expansion and maturation of DCs as evidenced by the expression of HLA-DR and CD11bCD11c surface molecules on the DCs generated from all groups. In conclusion, DCs can be ex vivo generated from control or patients with HCV or HCC in response to external stimulation. These results may be a promising tool for a therapeutic vaccine against HCV infection