Abstract: D-Lactic acid was produced with high purity by Leuconostoc pseudomesenteroides HL64. However, the key lactate dehydrogenase responsible for this biosynthesis remained undetermined. To address this, a systematic approach was implemented: Complete genome sequencing was conducted to annotate potential dehydrogenase genes, semiquantitative PCR was utilized to prioritize target candidates, and the putative D-lactate dehydrogenase gene was cloned in vitro, expressed heterologously, and purified for biochemical analysis. Enzymatic properties including substrate affinity and thermostability were characterized, establishing a foundation for subsequent strain engineering aimed at metabolic pathway optimization. The catalytic properties of LDH2 (OYT93_08575) were systematically characterized through biochemical assays. It was identified as encoding the key D-lactate dehydrogenase, with optimal reaction conditions determined to be pH8.0 and 30 ℃. Kinetic analysis revealed that the K_m values were calculated as 0.578 mmol/L for pyruvate and 0.275 mmol/L for NADH. Notably, catalytic constant (K_cat) of 45.04 s⁻¹ and catalytic efficiency (K_cat/K_m) of 7.88×10⁴ L/(mol·s) were observed for pyruvate, respectively, suggesting superior substrate affinity and turnover capacity. Substrate specificity profiling demonstrated that oxaloacetic acid and phenylpyruvate were also catalytically converted by this enzyme, though with lower efficiency compared to the primary substrate.