Abstract: Screened the fermentation medium for 9α-hydroxylation of androstenedione catalyzed by Nocardia canicruria BF313, and the reaction conditions, such as feeding mode, the initial pH were optimized, the fermentation medium was finalized:15g/L glucose, 2g/L peptone, 5g/L yeast extract, 1.5g/L FeSO4. Adjusted to pH7.0 before sterilization, then inoculated with 4% of the inoculum, added the substrate with Pseudo-crystallo mode after 16 h pre-culture. 48 h after conversion, the substrate conversion rate was 97.2% when the concentration of the feeding was 10g/L. Under the feeding concentration 20g/L, the substrate conversion rate could reach 82.7%.Conversion efficiency was much higher than the international level, so the industrial prospects would be great.