Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be one of the major public health problems in the world. The eventual control of this disease will require the development of a safe and effective vaccine. Bacille Calmette-Guerin (BCG), the only vaccine against TB, is not perfect for its limited ability to protect against the adult form of TB. Some improvements of TB vaccines relied to strengthening the immunogenicity and/or persistence of genetically modified recombinant BCG (rBCG) strain. Antigen 85B (Ag85B) and Mtb8.4 are importantly immunodominant antigens of M. tuberculosis, and both are very promising vaccine candidate molecules. MPT64(190-198), is presented to CD8(+) T cells during mycobacterial infections. In this study, we combined these above genes into one recombinant gene of ag85B-mpt64(190-198)-mtb8.4. Then we constructed the new rBCG containing this united gene. This rBCG can induce an increased Th1-type immune response in mice, characterized by an elevated level of interferon-gamma in antigen-stimulated splenocyte culture and a strong IgG2a antibody response. Also, it can elicit longer immune responses than BCG. The results show that this rBCG is a promising candidate for further study.