D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, K value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that ldLDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.