AbstractFermentation conditions for 2,3-butanediol (2,3-BD) production by Klebsiella pneumoniae CGMCC1.9131 were optimized statistically in shake flasks. Four significant factors including the initial concentrations of yeast extract, glucose, K2HPO4, and (NH4)2SO4 were optimized by Response Surface Methodology (RSM). To further improve the yield of 2,3-BD, EDTA Na2 was added to the medium. After optimization, the yield of 2,3-BD was 0.44 g/g glucose and the final concentration was 26.20 g/L when initial glucose concentration was 60 g/L. The enzymatic hydrolyzate of pretreated sugarcane bagasse by alkali-peracetic acid (PAA) and dilute acid were further used as feedstock to produce 2,3-BD under the optimized conditions, and the yields of 2,3-BD were 0.36 and 0.43 g/g consumed sugars, respectively. The experimental results indicated that the enzymatic hydrolyzate could be well converted to 2,3-BD.