Abstract: Objective To construct a targeting ribozyme (M1GS) which is specific to HCV genome. Methods According to the sequence of conservative sequence (5ˊUTR) of HCV genome, a guide sequence was designed and synthesized. And then by the PCR method, a kind of targeting ribozyme can be constructed by covalently linking the guide sequence to the 3ˊterminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. Results The engineered ribozyme（M1GS-HCV/C67） is targeted to the 5ˊUTR of HCV genome, and can effectively cleave the substrate RNA segment in vitro. The cleavage is specific and the cleavage site is between 67nt and 68nt of the target region. Conclusion The M1GS-HCV/C67 we got here would be a useful experimental material to further study its cleavage activity in vivo, and can be even used for evaluating its anti-viral effect in the animal model. It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications, and layed the foundation for developing a new nucleic acid drug of anti-HCV therapy.