Abstract: Cytochrome P450s enzyme in Streptomyces roseochromogenes SIIA-1902 was used to convert prednisolone into 16α-hydroxy prednisolone by electron transfer of spouse (roseoredoxin and roseoredoxin reductase). The results showed that peroxides could replace the natural oxygen reduction pathway in a cycle known as the ‘peroxide shunt’. In a NaIO4 supported peroxide shunt reaction, the yield of 16α-hydroxy prednisolone was 23.3 mg/L, and the conversion rate of prednisolone was 12.9%. Under the same conditions, in a hydrogen peroxide supported peroxide shunt reaction, the yield of 16α-hydroxy prednisolone increased to 45.3 mg/L, and the conversion rate reached 25.1%. The peroxide shunt reaction conditions were further optimized. When the concentration of prednisolone was 0.5 mmol/L, the mass fraction of hydroxypropyl cyclodextrin was 0.2 mg/mL, and the molar ratio of prednisolone to hydrogen peroxide was 1:3, the yield of 16α-hydroxy prednisolone was up to 64.3 mg/L and the conversion rate of prednisolone was 35.7% after 24 h of reaction at pH 7.2 and 30℃. Key words：P450s; prednisolone; peroxide shunt reaction