We constructed the DNA vaccine Ag85B-Rv3425 -Rv2029c-PPE26 (V569) plasmid based on p-VAX1-Ag85B-Rv3425-Rv2029c (A39) and evaluated its immunogenicity in mice. The levels of cellular immunity and humoral immunity against PBS, BCG, p-VAX1-Ag85B (A), p-VAX1-Ag85B-Rv3425 (A3), A39 and V569 were detected by enzyme-linked immunosorbent assay (ELISA) and flow cytometry after vaccination in mice. We found that V569 could trigger stronger Th1 immune response by augmenting the secretion of IFN-γ and significantly improve the CD4^＋/CD8^＋ T cell ratio in immunized mice compared to BCG. In addition, we evaluated the V569 as post-exposure DNA vaccine and found that it reduced bacterial burdens upon reactivation compared to BCG. The protection of V569 against latent TB infection was determined by zebrafish-Mycobacterium marinum latent infection model. In conclusion, V569 DNA vaccine may be a potential candidate DNA vaccine against latent tuberculosis infection.