L-Iactate oxidase (EC 1.1.3.X) belongs to a family of flavin mononucleotide (FNM) - dependent alpha-hydroxy acid-oxidizing enzymes that catalyses the oxidation of L-Iactate to form pyruvate and peroxide as end products. L-Iactate oxidase is not only used as markers in the production of wine, the food industry, but also clinical diagnostics, especially in the determination of blood lactate concentration to evaluate the respiratory status of the patient. LOX was found in a huge of yeast and bacterial strains, including lactic acid bacteria. In this paper, the gene encoding L-Iactate oxidase of Lactococcus lactis subsp. lactis VLSH-12 was amplified by PCR using a pair of primers LOXF and LOXR. Then PCR product was subsequently ligated into the TA cloning vector - pTZ57Rff and transformed into E. coli (TOPIOF' strain). The gene sequencing shows that the open frame reading for L-Iactare oxidase contained 1152 bp encoding for 383 amino acids. In order to produce LOX as extracellular enzyme in next step, the ORF was reconstituted with the promoter of a-amylase gene from B. subtilis 168M by megaprimer method using PlBs and LOXR as primers. New cassette was immediately cloned into pTZ57Rff and then ligated into the shuttle vector pHV33. The pHV33 carrying this cassette was transformed and expressed in E. coli (BL21(DE3). The recombinant protein showed activity at 63 Vlml which was higher than those of the wild-type ones. Protein purification was performed by chromatography system at 4°C. The purified protein showed molecular weight of 41 kDa.