Abstract: The influence of different carbon sources,nitrogen source,phosphate on 9α- OH- AD producted by Mycobacterium sp.BFZ304 was studied.On the base of single factors experiments,using 9α- OH- AD production as index response surface analysis was applied to optimize the fermentation medium composition. The quadratic regression analysis was applied to get the optimal level of main factors( corn steep liquor,glucose,Na NO₃、(NH₄)_2HPO₄).Finally determined the optimal medium were as follows: corn steep liquor 40 g /L,glucose 10 g /L,Na NO₃ 6 g / L,( NH₄)2HPO₄ 0.7 g / L.Under these optimal conditions,the production of the 9α- OH- AD was high up to 4.86 g / L when the concentration of the feeding was 10 g / L,increased by 40% compared with the original medium.It would have a disirable prosfect.