Optimization of the fermentation medium for DDase production by Gluconaobacter oxydans M5 was carried out in the shake flasks using two kinds of statistical methods. Four variables, namely glucose, tryptone, yeast extract and sodium chloride, were found to influence DDase production significantly by the Plackett-Burman screening. A four-factor five-level central composite design (CCD) was chosen to explain the combined effects of the four medium constituents. The optimum medium consisted of glucose (17.670 g/L), maltobiose (30 g/L), tryptone (12.198 g/L), yeast extract (13.528 g/L), ammonium nitrate (15 g/L), copper sulfate (0.01 g/L), zinc sulfate (0.01 g/L), and sodium chloride (0.009 g/L); the initial pH 6.0 was set prior to sterilization. The DDase yield obtained from optimized medium increased by 17-fold (0.238 U/mL) or so. Under these optimal conditions, the experimental values agreed with the predicted values, indicating that the chosen method of optimization of medium composition was efficient, relatively simple, time reducing and material saving.