The genes encoding 19kD lipoprotein and early secreting antigenic target-6( ESAT-6) were amplified from genome of the standard Mycobacterium tuberculosis H37Rv using polymerase chain reaction, and then cloned into the vector pMD18-T followed by subcloning into the expression vector pET-21a, respectively. Recombinant 19kD-ESAT6 was expressed in E. coli, and then purified by Ni-NTA. The antigenicity of the fusion protein was analyzed by Western blot analysis. The recombinant 19kD-ESAT6 plasmid was constructed successfully, and could be expressed efficiently in E. coli BL21 ( DE3) . The relative molecular mass of the fusion protein was approximately 29 ×10³ by SDS-PAGE. The recombinant 19kD-ESAT6 protein showed specific antigenicity as determined by Western blot, and can be used for the development of serodiagnostic reagents.