3-Ketosteroid-9 alpha-hydroxylase (KSH) consists of two protein systems, KshA and KshB, and is a key enzyme in microbial degradation pathway of natural sterols. 9 alpha-Hydroxy-4-androstene-3,17-dione (9 alpha-OH-AD) is a valuable steroid pharmaceutical intermediate. The expression of a 3-ketosteroid-9 alpha-hydroxylase oxygenase (KshA1) with a broad substrate range and high hydroxylation ability was enhanced in Mycobacterium sp. LY-1 to improve the yield of 9 alpha-OH-AD. Through whole-genome sequence mining and homologous comparison, the putative genes (kshA1 and kshB) in wild strain LY-1 were firstly identified. Then they were heterogeneously co-expressed in Escherichia coli BL21. The transformation results of recombinant BL21-KshA1/B demonstrated KshA1/B had high hydroxylation ability to AD. Moreover, substrate preference analysis suggested that KshA1(LY-1) had a broad substrate range. After enhancing expression of kshA1 and kshB in the strain LY-1, the maximum productivity of 9 alpha-OH-AD in recombinant LY-1-KshA1/B reached 0.064g/L/h in a 5-L stirred fermenter.