Hepatitis C virus (HCV) infection is a major risk factor of chronic hepatitis, liver cirrhosis and even hepatocellular carcinoma (HCC). However, the detailed mechanism on cancer caused by HCV is still elusive. Nonstructural protein 5B (NS5B) is one of the nonstructural proteins encoded by HCV, and essential for HCV RNA replication because of its RNA-dependent RNA polymerase (RdRp) activity. In addition to participating in viral RNA replication, NS5B was also found to be involved in the pathogenicity of HCV through direct interaction with host proteins. In order to address the role of NS5B in HCV replication and pathogenicity, we constructed a tetracycline-inducible (Tet-on) stable cell line expressing NS5B in HepG2 cells. Protein expression was examined by Western blot using both tag antibody and anti-NS5B antibody induced by doxycycline either at different concentrations or at different indicated time points. Moreover, immunofluorescence assay further confirmed the results. In conclusion, the HepG2 Tet-on NS5B cell line will be a good model to investigate the role of NS5B in HCV replication and pathogenicity.