Tuberculosis is a serious infectious disease caused by Mycobacterium tuberculosis. DNA vaccination is an advanced technique for protecting human bodies from infectious diseases including tuberculosis by injecting exogenous gene engineering DNA into the body to produce an immunological response. This research mainly focused on the immunogenicity of new DNAvaccines: pVAX1-64. Recombinant vector pVAX1-64 was constructed, identified successfully and then transfected into BHK-21 cells and screened of stable expressing cell lines. Specific antibody titer test in serumof immunizationmice and tests for allergic reaction in immunization guinea pigswere performed. FinallyCD4+ÂACD8+ T cells in immunization micewere detected. The antibody titer ofMPB in the immunizedmice serum was obviously increased significantly with the increased immune days(p<0.01). Compared with saline group and pVAX1 group, there were significant increased on the number of CD4+ and CD8+ T cells in BCG and pVAX1-64 group (p<0.05). There was no allergic reaction after the immunization in guinea pigs, which were sensitive to mycobacteria, using the recombinant expression plasmid pVAX1-64. This new DNAvaccines would not interference the PPD test. All these suggested that this new DNA vaccines is an ideal vaccine and may be further developed as a useful method to prevent tuberculosis.