Vitamin C precusor--2-keto-L-gluconic acid can be prepared directly by mixed culture of Gluconobacter oxydans SCB329 and Gluconobacter suboxydans SCB110. To obtained its high yield, firstly, the proportion of the two microorganisms, the ingredients of medium and the initial pH were optimized in shake flasked, then L_(9) (3~(4)) orthogonal experiment confirmed that urea, C. S. L had high degree statistical meaning. Based on these data, an optimized fermentation media was obta ined: D-Sorbitol 9g, C. S. L1.5g, KH_(2)PO_(4) 0.1g, CaCO_(3) 0.2g. By-product can be inhabited to the greatest extent and the yield increases by 20%.