Abstract: Objective In order to obtain the large amount of M. tuberculosis Ag85A protein by prokaryotic expression. Methods The fbpA gene encoding M. tuberculosis Ag85A protein was amplified by using polymerase chain reaction (PCR) from M. tuberculosis H37RV strain. The PCR product was cloned into prokaryotic expression vector pProEXHTb to generate the recombinant plasmid pProfbpA, which was then transformed into the competence Escherichia coli BL21 cells. The recombinant Ag85A protein was successfully expressed by being induced with isopropyl thio-β-D-galactoside (IPTG) and purified by the Ni-Purification System. The distribution of fbpA gene in different environmental mycobacterial strains was screened by PCR and ELISA was performed to determine the immunoreactivity of the recombinant Ag85A protein with sera from different mycobacterial infections. Results 32 kd Ag85A protein was successfully expressed and purified. It was confirmed by PCR and ELISA that fbpA gene presented in the genomes of M.tuberculosis H37Rv, H37Ra, BCG, M. smegmatis, M. terra, M. trivial and M. phlei, but being absent in the genomes of M. vaccae. There were the highest Ag85A antibody titers in serum of TB patients and mice which being infected by M. tuberculosis H37Rv. Conclusion The recombinant Ag85A protein was successfully expressed and purified.