The purpose of the study is to clone, identify, and express the mature secreted protein MPB63 from Mycobacterium bovis(MB) and to play a foundation for diagnosis of MB, for applying MB vaccine into clinic practice, and for detecting of immunity effectiveness. The gene encoding MPB63 was amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, PCR product was approximately 400 bp. Clone vector pGEM-T-63 was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique. pGEM-T-63 and pET28a(+) were digested by BamHⅠ and EcoRⅠ double enzymes. The porkaryotic expression vector pET28a-63 was constructed by using the purified MPB63 gene that was subcloned into the expression vector pET28a(+). Plasmid containing pET28a-63 was transformed into competence E.coli BL21 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 18ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting and it had antigenic activity of MB. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.