Abstract： AIM: To construct a eukaryotic expression vector with dual-target ribozyme against hepatitis B virus and to observe the inhibition of HBeAg expressed in the HHCC cell (human hepatic carcinoma cell line) transfected by HBV genome and dual-target ribozyme against HBV. METHODS: Using subclone technique, the dual-target ribozyme gene cut from pGEM-Rz 123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid pBBS212-Rz and p1.2Ⅱ carrying genome of adr-subtype HBV were cotransfected into the HHCC cell by using lipofectamine. The intracellular e/c antigen of HBV was detected by ELISA and immunohistochemistry. RESULTS: The cotransfected cells did express HBV e antigen and ribozyme RNA, and the dual-target ribozyme inhibited the level of HBeAg expressed on cotransfected cells by up to 65%. CONCLUSION: The dual-target ribozyme can cleave the HBV core region mRNA and inhibit HBV the expression of HBV core region gene.