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<i>Plasmodium knowlesi</i> is the only <i>Plasmodium</i> that causes zoonotic disease among the <i>Plasmodium</i> that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by <i>Plasmodium</i> spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of <i>P. knowlesi</i> LDH (PkLDH) using a bacterial expression system for studying malaria caused by <i>P. knowlesi</i>. Recombinant pET-21a(+)-<i>PkLDH</i> plasmid was constructed by inserting the <i>PkLDH</i> gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing <i>Escherichia coli</i> Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by <i>P. knowlesi</i>.