It is estimated that one in four people worldwide carries Mycobacterium tuberculosis bacteria. MPT64 is a protein exclusively secreted by Mycobacterium tuberculosis complex (MTC) bacteria. It serves as a crucial diagnostic marker and plays a role in the bacterium’s survival by modulating the host immune response. Consequently, the development of innovative diagnostic tools based on MPT64, as well as the production of high-purity MPT64 protein to support research on tuberculosis pathogenesis and the advancement of novel therapeutic strategies, is of great importance. In this study, optimization experiments were conducted to produce this protein in E. coli with high yield and purity. First, a gBlock was designed by codon optimization and then cloned into a plasmid vector using the LIC method. For more efficient production, E. coli BL21(DE3)-R3-pRARE2 strain, which carries rare tRNAs for rare codons, was used as the host. Five different culture media were tested to maximize protein production, with the highest yield obtained in eBHI medium. The resulting protein yield was 4.9 mg/L. To the best of our knowledge, this study provides the most detailed information on the recombinant production and characterization of MPT64 to date. Therefore, these results contribute important data for future studies on the MPT64 protein.