Background: Vaccination is one of the effective measures to prevent latent tuberculosis infection (LTBI) from developing into active tuberculosis (TB). Applying bioinformatics methods to pre-evaluate the biological characteristics and immunogenicity of vaccines can improve the efficiency of vaccine development. Objectives: To evaluate the immunogenicity of tuberculosis vaccine W541 and explore the application of bioinformatics technology in tuberculosis vaccine research. Methods: This study concatenated the immunodominant sequences of Ag85A, Ag85B, Rv3407, and Rv1733c to construct the W541 DNA vaccine. Then, bioinformatics methods were used to analyze the physicochemical properties, antigenicity, allergenicity, toxicity, and population coverage of the vaccine, identify its epitopes, and perform molecular docking with MHC alleles and Toll-like receptor 4 (TLR4) of the host. Finally, the immunogenicity of the vaccine was evaluated through animal experiments. Results: the W541 vaccine protein is a soluble cytoplasmic protein with a half-life of 1.1 hours in vivo and an instability index of 45.37. It has good antigenicity and wide population coverage without allergenicity and toxicity. It contains 138 HTL epitopes, 73 CTL epitopes, 8 linear and 14 discontinuous epitopes of B cells, and a strong affinity for TLR4. Immune simulations showed it could effectively stimulate innate and adaptive immune responses. Animal experiments have confirmed that the W541 DNA vaccine could effectively activate the Th1- and Th17-type immune responses, producing high levels of IFN-γ and IL-17A, but could not significantly increase antibody levels. Conclusion: the W541 DNA vaccine can induce strong cellular immune responses. However, further optimization of the vaccine design is needed to make the expressed protein more stable in vivo. Bioinformatics analysis could reveal vaccines’ physicochemical and immunological information, which is critical for guiding vaccine design and development.