Objective To study immunogenicity of fusion gene Ag85B-Esat6-HspX of Mycobacterium tuberculosis． Methods Forty BALB / c mice were randomly divided into four groups (10 per group) : the plasmid pcDNAHspX group，the pcDNA-Ag85B-Esat6-HspX plasmid group，the BCG group and the saline group． The immunization was repeated three times two weeks apart． A single dose of BCG (1 × 106 CFU /mouse) was injected subcutaneously． Mice in other three groups were administered intramuscularly in the hind limb． Total IgG levels were detected at 2，4 and 6 weeks after immunization． Meanwhile，cell-mediated immunity responses were examined two weeks after the final immunization as well． Result Mice immunized with recombinant plasmid expressing the Ag85B-Esat6-HspX fusion protein generated specific cellular and humoral immunologic response to the stimulation of Ag85B，Esat6 and HspX． Conclusion The fusion gene Ag85B-Esat6-HspX of Mycobacterium tuberculosis could be DNA vaccine to carry out the further research of protective efficacy．