For the purpose of producing pyruvate from dl-lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production,was chosen for further research. Physiological studies and 16S rDNArelationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39℃, respectively. Low-concentration EDTA (1mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. Vmax and Km value for dl-lactate were 2.46 mol/(min mg) protein and 9.53mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300mg/l of crude enzyme (4037U/ml lactate oxidase), could convert 66% of 116mM of dl-lactate into 76.6mM pyruvate in 18h, and 82% of substrate was transformed after 48 h, giving 95.0mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).