The gene encoding ESAT-6 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR), the PCR product was approximately 288bp DNA segment. The PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-ESAT-6 was constructed successfully. The purified ESAT-6 gene was subcloned into the expression vector pGEX-4T-3, and the prokaryotic expression Plasmid pGEX-4T-3-ESAT-6 was constructed. Plasmid containing pGEX-4T-3-ESAT-6 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-beta-D- thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 34 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity of M. bovis . The results were expected to lay foundation for further studies on the subunit vaccine, DNA vaccine and diagnostic reagents of ESAT-6 gene in their prevention against bovine tuberculosis.