Abstract: 9α-hydroxy-4-androstene-3, 17-dione (9α-OH-AD) was an irreplaceable intermediate of steroid drugs, which was an important raw material for the synthesis of many corticosteroid drugs. In this study, the 3-sterone-9α-hydroxylase (KSH) gene kshA and kshB were successfully expressed in androster-4-ene-3, 17-dione (AD) producing strain Mycobacterium sp.TFZ, and a 9α-OH-AD engineering strain was obtained. The yield of 9α-OH-AD was used as an evaluation index to optimize the type and content of emulsifier Tween in the oil-water two-phase conversion system. The results showed that, compared with the starting strain Mycobacterium sp.TFZ, the content of 9α-OH-AD in the fermentation broth of the Mycobacterium sp.TFZ3215 increased from 1.7% to 94.7%, and the AD contents decreased from 80.1% to 3.4%. After adding 2 g/L of Tween-80 to the oil-water two-phase fermentation system, the yield of 9α-OH-AD increased from 1.69 g/L to 7.53 g/L, which increased by 3.4 times. In this study, a high-yield 9α-OH-AD engineering strain was successfully constructed, and an oil-water two-phase fermentation system was optimized. The yield of 9α-OH-AD reached 7.53 g/L, which had excellent industrialization prospect.