The invention discloses a method using enzymatic-method conversion to produce 9alpha-hydroxyandrost-4-ene-3,17-dione and belongs to the field of gene engineering and enzyme engineering. The method hasthe advantages that the oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-sterone 9alpha-hydroxylase from Mycobacterium sp. Strain VKMAc-1817D are successfully expressed in E. coliBL21, and the fact that the KshC is an oxidation subunit, and the enzyme activity of the KshC is much higher than the KshA is authenticated; BL21/pET-28a(+)-fdh built in a lab is used to express formate dehydrogenase FDH, the crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria are used as the biological catalyst, a steroidal compound AD is used as the substrate, optimal reaction temperature is determined as 30 DEG C, and pH is 7.0; the AD is converted under the optimal conditions to generate the product 9-OH-AD, the yield of the 9-OH-AD within 20 hours is 4.7g/L,and mole conversion rate reaches 96.7%; coupling of a 3-sterone 9alpha-hydroxylase hydroxylation system and a coenzyme circulating system is achieved by using the method to produce the 9-OH-AD, and the method is high in efficiency, low in cost, green, environmentally friendly and the like.