Objective:Rabies virus glycoprotein (RVG) was cloned using Bac-to-Bac baculovirus expression vector system and expressed in Spodoptera frugiperda (Sf-9) cells. Furthermore, the purified r-RVG protein was used to evaluated the immunological characteristics. Methods: Referring to the sequence of G protein gene of rabies virus CVS-11 strain from GenBank, we designed a pair of specific primers for PCR amplification, cDNA worked as the PCR template. The sequence of G protein gene was amplified by specific primers designed according to the CVS-11 strain from GenBank. The PCR-amplified RVG gene was cloned into the pFastBac-GP67B plasmids with digestion by restriction enzyme BamH I and Kpn I. Positive clone was transformed into E.coli DH10Bac competent cells and then the recombinant rBacmid-RVG was identified by blue /white selection and PCR analysis. The recombinant baculovirus was generated by transfecting Sf-9 cells and the recombiant RVG from eukaryotic expression was purified with His-Trap, characterized by SDS-PAGE and Western-blot analysis. The immunogenicity and serum neutralizing activity of recombiant RVG were assessed using mice model. Results: Recombiant RVG protein was efficiently expressed in eukaryotic expression and purified, with molecular weight of 58 000. The mice serum showed neutralizing activity after immunization by recombiant RVG. Conclusion: The results of the study indicated that recombiant RVG retained the biological activity as the native conformation, thereby paving the way for producing efficacious subunit vaccine and screen neutralizing antibodies in future research.