Hepatitis C virus(HCV) encoded non-structure protein 5B(NS5B) is believed to be a RNA dependent RNA polymerase. GST-NS5B fusion protein was expressed and purified and its ability to bind to the 1～585 nucleotides of 3′-terminal negative-strand RNA sequences was examined by UV cross-linking. Results presented here demonstrated that the NS5B binding to this region increased with the dosage of protein. The binding ability of NS5B to 3′-terminal negative-strand RNA sequences was approximately 10 folds higher than to 3′ UTR X region of positive-strand RNA. The specificity of NS5B binding to 3′-terminal negative-strand RNA sequences was tested by competition with unlabelled RNA probe or an unrelated RNA/proteins. Results showed that the excess amount of cold probe RNA was able to almost completely compete out the complex resulted from protein-RNA interaction. However unrelated RNA and protein were demonstrated no competition with NS5B. These results suggest that NS5B is a participating component of 3′-terminal replica of HCV negative-strand RNA.