The Mycobacterium tuberculosis DNA vaccine pVAX1/Ag85A­Rv3425­Rv2029c (A39) plasmid was constructed and its immunogenicity was then investigated. Ag85A gene was firstly amplified by polymerase chain reaction (PCR) and then cloned into pVAX1 to construct the recombinant plasmid pVAX1/Ag85A (A). Ag85A­Rv3425 gene segment was amplified by PCR and then cloned into pVAX1 to construct the recombinant plasmid pVAX1/Ag85A­Rv3425 (A3). Rv2029c gene was amplified by PCR and then cloned into plasmid A3 to construct the recombinant plasmid A39. HEK293T cells were transfected with the recombinant plasmids and the protein expression was detected by Western blotting. The proteins Ag85A, Rv3425 and Rv2029c were expressed in Escherichia coli BL21. C57BL/6 mice were divided into five groups named phosphate buffered saline (PBS), pVAX1, A, A3 and A39. The mice were immunized with plasmids mediated by in vivo electroporation (EP) 3 times at an interval of 2 weeks. The levels of cellular immunity and humoral immunity were detected by enzyme­linked immunospot assay (ELISPOT), enzyme­linked immunosorbent assay (ELISA) and flow cytometry. In the immunized mice, A39 could cause stronger cellular immunity: the high­level secretion of IFN­γ， IL­2 and TNF­α， and the increase in the proportion of CD4^＋/CD8^＋ T cells and CD8^＋  perforin^＋ T cells. Therefore, the constructed A39 DNA vaccine could induce stronger cellular immunity in mice and it may be used as a new tuberculosis vaccine against latent tuberculosis infection.