Abstract: Single factor experiment, orthogonal test and response surface analysis were used to determine the optimal medium components and fermentation condition available for L.mesenteroides 6055 to synthesize glucooligosaccharides.The results showed that the optimum media favoring glucooligosaccharides formation were: sucrose ( 100g /L) , maltose ( 50g /L) , yeast extract ( 5g /L) , K2HPO4 ( 3g /L) , and Tween 80 ( 2mL) .The optimum fermentation condition for L.mesenteroides 6055 was inoculum of 3.0%, 25.0℃, and pH7.10, and the yields of glucooligosaccharides reached 5.15g /L.Addition of 2% Saccharomyces cerevisiae to fermentation liquor is able to metabolize selectively 100% fructose and glucose, and 92.8% maltose, the proportion of glucooligosaccharides formed by L.mesenteroides 6055 increased from 37.5% to 74.3%. And the glucooligosaccharides produced by L.mesenteroides 6055 whose concentration is 2% promoted proliferation of gastro- intestinal probiotic strains such as Bifidobacterium animalis subsp.lactics Bb-02 and Lactobacillus acidophilus NCFM by 37.9% and 48.6%, respectively.