A lactate oxidase was purified about 36-fold from a newly screened strain KY6 of gram negative bacterium from soil to yield a homogeneous protein. The native enzyme had a molecular mass of 204 kDa easured by Sephadex G-200 and that of subunit on the SDS-PAGE was found to be 45 kDa. The enzyme was optimally active at pH 7.7 and showed stability at pH range of 5.7 to 9.5 for 24 h at 4℃. The optimum temperature was 70℃ and the enzyme activity was stable for 10min up to 45℃. The half-life of the enzyme activity was about 10 min at 55℃. The best substrate of the enzyme was D-lactate and Km value for D-lactate was 0.14raM. The Km value for DL-lactate was 0.20mM. Substrate inhibition of the enzyme was observed at higher concentrations than 20mM of DL-lactate and 10mM of D-lactate.