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% $Na_2S_2O_3$, 0.2% $KH_2PO_4$, 0.2% $K_2HPO_4$, 0.04% $Na_2CO_3$, 0.02% $MgSO_4$.$7H_2O$, 2ml trace elements solution, ann PH 6.5 at 3$0^{circ}C$ and 72hr cultivation. The oxidase was successively purified 83 folds yield by ${(NH_2)}_2SO_4$ 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 40t and PH 7.0 The enzyme activity was relatively high by $eta$-mercaptoethanol but strongly inhibited by cysteine. Key Words : thiobacillus neapolitanus