The identification of a large number of antigens with potential for development of new tuberculosis vaccine has been accomplished in recent years. This study was designed for cloning and expression of ESAT-6 as a potent antigen of Mycobacterium tuberculosis. Selected gene (Rv3875) was amplified by PCR and product was ligated into expressing plasmid vector pQE30 and recombinant pQE30-ES plasmid was constructed. This hybrid vector was transformed in E. coli M15 and expressed in optimal condition. The expressed protein was analyzed on SDS-PAGE and confirmed by western blotting using specific antisera to ESAT-6. We successfully cloned and expressed ESAT-6 (His)₆ from M. tuberculosis H37Rv genome. As well as usage for serodiagnosis, this recombinant protein offers the potential development of other vaccine formats such as DNA or subunit vaccines against tuberculosis.