The invention relates to a tuberculosis gene vaccine assembled by a chitosan delivery system, which consists of chitosan and tuberculosis antigen encoding plasmids, wherein full-length genes of tubercle bacillus heat shock proteins HSP65 are inserted into the tuberculosis antigen encoding plasmids; and four T cell epitope genes EAST-6[189-228], Ag85A[369-405], CFP10[162-207] and Ag85B[420-459] from tubercle bacillus antigens are inserted into the HSP65 full-length genes. The invention also discloses application of the tuberculosis gene vaccine. By performing nasal drip of the gene vaccine on an immune mouse, the gene vaccine is proved to be capable of inducing response to special antibodies of a plurality of tuberculosis antigens, inducing killing response of locally strong tuberculosis special T cells of the whole body and lung mucous membranes, simultaneously inducing immunological response of Th1 which secretes high-level IFN gamma and have the effect which is obviously superior to the prior BCG vaccine, and is a superior vaccine for preventing and treating tuberculosis.