Thiobacillus neopolitanus R-10, which produces a active thiosulfate oxidase, was isolated from nightsoil. The optimal culture conditions of Thiobacillus neopolitanus R-10 for the production of enzyme was determined as followed: 0.8% Na2S203, 0.2% KH2P04, 0.2% K2HP04, 0.04% Na2C03, 0.02% MgSO4·7H2O, 2ml trace elements solution, and pH 6.5 at 30℃ and 72hr cultivation. The oxidase was successively purified 83 folds yield by (NH2)2S04 fractionation, DEAE-Cellulose, Sephadex A-50 column chromatogrophy and gel Sephadex G-150 gel filteration with yield of 5.9%. The molecular weight of purified enzyme was estimated to be 43.000 dalton by SDS-polyacrylamide gel electrophoresis and gel filteration column chromatography. The enzyme activity was highest at 40℃ and pH 7.0 The enzyme activity was relatively high by β-mercaptoethanol but strongly inhibited by cysteine.