Abstract: Combined action with multiple enzymes was investigated for isomaltooligosaccharides( IMOs) production in this study. The corn starch was liquefied using thermostable α- amylase,followed by simultaneous saccharification and transglycosylation using α- glucosyltransferase,pullulanase and β- amylase. The reaction condition was optimized through single factor experiments,including liquefaction degree,reaction temperature and p H in saccharification and transglycosylation phase,α- glucosyltransferase dosage,pullulanase and β- amylase dosage in saccharification phase.The optimum reaction condition was obtained when the dextrose equivalent value( DE) was control at 20~30 and 500~1000 U/g of α- glucosyltransferase,0.9 U/g of pullulanase and 500 U/g ofβ- amylase were employed for simultaneous saccharification and transglycosylation at p H6.0 and 55 ℃ with the25% starch slurry. Under this optimal condition,IMOs with 49.09% of isomaltose,isomaltotriose and panol was obtained within 15 h incubation.In conclusion,the new developed procedure processed rapid preparation of IMOs with high efficiency,in which the IMOs index was significantly improved and the manufacturing duration was 70%reduced.The results are able to improve or reform the current industrial scale process for IMOs manufacturing.