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Aim: To construct the HCV response minigenome harboring IFN-β, and investigate its inhibitory effect on HCV RNA replication.Methods: First, we extract lymphocytes from peripheral blood and stimulated by poly I:C to express the IFN gene.Total RNA extracted from lymphocytes was reversely transcripted into cDNA.IFN-β gene was amplified by PCR and was sequenced, and then the correct gene was cloned into pcDNA/5UrI3URz in antisense direction to obtain pcDNA/5UrIFNI3URz.The plasmid was linearized by EcoR I and transcripted into RNA in vitro, then transfected into Huh7.5BB7.After 48hrs, cell was harvested and total proteins were extracted to do Western-blot, IFN-β antibody as the first antibody.Results: Western-blot analysis showed that IFN-β could be specifically expressed after the pcDNA-5UrlFNI3URz was trasnfected into Huh7.5BB7 cell lines.The real-time PCR assay showed that the level of HCV RNA replication was distinctly decreased, and viral NS3 protein level was reduced compared with pcDNA alone.Conclusion: The HCV minigenome expressing IFN-β can efficiently block the viral replication.This study may provide a new strategy against HCV infection.