Antigen 85B (Ag85B) and ESAT-6 are important immunodominant antigens of Mycobacterium tuberculosis, and both are very promising vaccine candidate molecules. In this study, we relied on the T-cell epitopes of Ag85B and ESAT-6 to design a chimaeric protein by inserting ESAT-6 into Ag85B from the amino acids 167-182. We found the ratio of IgG2b/IgG1 and the secretion of interferon (IFN)-gamma in the mice vaccinated with the new protein with adjuvant MPL and TDM were higher than the mice immunized with fusion protein Ag85B-ESAT-6, which have been reported and could induce levels of protective immunity similar to BCG in the mouse model of tuberculosis (TB) infection. These results suggest that the chimaeric protein Ag85B(N)-ESAT-6-Ag85B(C) is a strong candidate for further study and the T-cell epitopes of the antigens should be considered when we design the subunit vaccine.