The invention relates to a preparation method of isomaltooligosacharide. The preparation method comprises the following steps of 1, adding water into a starch raw material, adjusting a concentration and a pH value of the slurry, and adding high temperature-resistant alpha-amylase into the slurry to obtain starch slurry, 2, carrying out liquidation on the starch slurry to obtain a liquefied liquid, 3, adding maltotriose generation enzyme (AMANO AMT1.2L) and pullulanase (GENENCOR OPTIMAX L-1000) into the liquefied liquid, carrying out thermal-insulation saccharification, and carrying out enzyme denaturalixation to obtain a saccharified liquid, 4, adding alpha-glucosidetransferase into the saccharified liquid for a reaction to produce a primary trans-glucoside saccharified liquid, adding alpha-glucosidetransferase into the primary trans-glucoside saccharified liquid for a reaction, and carrying out enzyme denaturalixation to obtain a trans-glucoside saccharified liquid, and 5, carrying out decoloration on the trans-glucoside saccharified liquid, carrying out filtration, carrying out ion exchange, carrying out chromatographic separation, carrying out concentration and carrying out drying to obtain isomaltooligosacharide. The preparation method greatly improves contents of sugars such as panose, isomaltotriose and tetrasaccharide having polymerization degrees greater than or equal to 3 in the reaction product liquid.