Abstract: Objective: To investigate the gene expression of non-structural protein 4B （NS4B） of hepatitis C virus （HCV） in yeast. Methods: Polymerase chain reaction （PCR） was performed to amplify the gene of HCV NS4B from the plasmid pBRTM/HCV-1 containing the whole fragment of HCV and the gene was cloned into pGEM T vector. The gene of HCV NS4B was cut from the recombinant pGEM T-NS4B plasmid and cloned into yeast expression plasmid pGBKT7, then pGBKT7-NS4B was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting analysis. Results: HCV NS4B gene was successfully cloned into pGBKT7. The results of SDS-PAGE and Western blotting assay indicated that the relative molecular weight of the expressed product was approximately 48 ku and HCV NS4B protein existed within yeast cells. Conclusion: The findings suggested that HCV NS4B protein was successfully expressed in yeast system.