Objective To determine the expression of recombinant Mycobacterium tuberculosis (MTB) specific antigen ESXO in E.coli, and evaluate its immunogenicity. Methods Recombinant ESXO protein of MTB was obtained with conventional molecular cloning method. The serum antibody against ESXO was detected by enzyme linked immunosorbent assay (ELISA) in 84 patients with tuberculosis (TB) and 48 healthy controls, the serum immunogenicity was evaluated, and the results were compared with findings in detection of ESAT-6 and CFP-10 protein for serodiagnosis of TB. Results The recombinant specific antigen ESXO was expressed as soluble and inclusion body protein in E.coli BL21 plysS (DE3), with the relative molecular weight of 27 300, which was in line with expectations. The immunogenic evaluation of recombinant ESXO protein indicated that its specificity and sensitivity reached 94.0% (45/48) and 32.1% (27/84) respectively, which was equal to ESAT-6 and CFP-10 in specificity and higher than ESAT-6 in sensitivity. Conclusion The specific antigen ESXO is expected to be a novel candidate antigen for the diagnosis and vaccine design in TB.