To enhance immunogenicity of HIV-1 cross neutralizing epitopes , three HIV-1 cross neutralizing epitopes (ELDKWA, NWFDIT, GPGRAFY) were fused to 3' end of HBV S gene by PCR cloning technology, respectively. Three vaccinia virus (Tiantan strain) recombinants expressing separately the three fusion genes were subsequently constructed, named as RVJ1175S-2F5 (ELDKWA), RVJ1175S-4E10 (NWFDIT) and RVJ1175S-447-52D (GPGRAFY), respectively. From the supernatants of CEF cells infected by these vaccinia recombinants, three subunit vaccines (PS-2F5, PS-4E10 and PS-447-52D) were prepared after purification. Biology and immunology characteristics of these fusion antigens in vaccinia recombinants and subunit vaccines were comparatively studied. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus (Tiantan strain) correctly. The Fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two bands were reacted well to HBsAb and corresponding HIV-1 monoclonal antibodies 2F5, 4E10 and 447-52D. BALB/c mice were immunized with subunit and vaccinia recombinant vaccines by intraperitoneal injection. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope antibody in peripheral blood of immunized mice were tested by ELISA, and all the antibody titers induced by three subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. This work provides a basis for future study on neutralizing activity of these immunized sera and enhancing immune effect through the combined immunization with different type of vaccines.