Abstract: Removing the glucose from malt syrup could disarm the substrate inhibition and improve the trehalose production by enzymatic method.In this study,the removing efficiency of glucose from malt syrup was improved by yeast fermentation,and the fermentation parameters was optimized by mono-factor analysis and orthogonal test.Results showed that at the presence of 40% malt syrup,4% yeast,99.4% glucose could be removed from malt syrup after incubation for 6 hour at 32 ℃ p H5.5. As a consequence,the trehalose yield increased from 29.8% to58.4% which was a 96.0% increase compared with that obtained before. Our results provide a promising strategy for industrial production of trehalose using malt syrup as substrate.