Background and Aim: Malate dehydrogenases (MDH, L-malate: NAD oxidoreductase, E.C.۱.۱.۱.۳۷), catalyzes the NAD+/NADH-dependent interconversion of the substrates malate and oxaloacetate. MDH plays a key role in the tricarboxylic acid cycle. The bacterial MDH is a dimeric enzyme with a molecular mass of ۷۰ kDa. This enzyme is used in the aspartate transaminase diagnostic kit that used in the diagnosis of liver disease. Due to its important it was decided to clone and express this protein.Methods: The nucleotide sequence of the malate dehydrogenase was optimized, synthetized and then inserted into cloning vector, pUC۵۷. The gene was then cloned into an expression vector, pET-۲۸a⁺, between the two restriction sites of Nde I and Xho I. The recombinant vector was transformed into E.coli strain BL۲۱ (DE۳) and its expression was examined using different conditions. The protein was expressed in LB medium for ۳h, ۶h, ۱۵h and ۲۱h at ۳۷°C after inducing by ۰.۴ mM and ۱ mM of isopropyl β-D-۱-hiogalactopyranoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the optimal condition.Results: The constructed expression vector, pET۲۸ – malate dehydrogenase, was transformed into E. coli BL۲۱ (DE۳) and its cloning was confirmed by sequencing. Following expressing the enzyme in different conditions, SDS-PAGE illustrated that the best protein expression is achieved in LB medium at a concentration of ۰.۴ mM IPTG and induction for ۱۵ h at ۳۷°C. In the above conditions, a soluble and active product was obtained. The kinetics and stability of this enzyme in the presence of various additives will be investigated in future.Conclusion: In this study we have firstly cloned and then expressed the recombinant MDH enzyme using pET-۲۸ expressing system. The product obtained in this process was soluble and active. This protein can used in production of diagnostic kits, in the future.