The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH 4 ) 2 SO 4 , MgSO 4 ·7H 2 O and initial pH on chitosanase production was noted with Plackett–Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0g/L (NH 4 ) 2 SO 4 , 1.3g/L MgSO 4 and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6U/mL to 118U/mL.