Abstract: Mycobacterium bovis is the etiological agent of bovine tuberculosis (BT). The secretory proteins Ag85B, MPB64 and ESAT-6 are the major immunogenic antigens of M. bovis, and they play important roles in inducing immune responses that confer resistance against infection. In the present study, using pcDNA3.1(+) as vector, various DNA vaccines were constructed with the genes encoding the three antigens as follows: fusion of three genes (pcDNAMPB64/Ag85B/ESAT-6, pcDNA-MAE) and trivalent combinations (pcDNA-Ag85B + pcDNA-MPB64 + pcDNA-ESAT-6). The immune efficacy of the DNA vaccines were evaluated based on serum antibody titers, lymphocyte proliferation assay and content of cytokine (interferon-γ and interleukin-2)titers. Protective efficacy following challenge with M. bovis Bacille Calmette-Guérin (BCG) was evaluated based on lung tissue bacterial loads and histopathologic changes. The data demonstrated that immunization with fusion DNA vaccines (three genes) resulted in significantly higher serum antibody levels, lymphocyte proliferation (SI) values, IFN-γ and IL-2 levels than immunization with polyvalent DNA vaccines (P <0.05). Additionally, fusion DNA vaccines provided superior protection than polyvalent DNA vaccines following BCG challenge. The protective efficacy of the fusion DNA vaccines was equivalent to that of the BCG vaccine, suggesting that fusion DNA vaccines provide a promising approach for the prevention of BT.