Abstract: With the acorn starch as raw material, the process of preparation of isomalto-oligosaccharide by enzymatic methods were optimized in order to increase the content of isomaltose, panose and isomaltotriose of isomalto-oligosaccharide. Box-Benhnken response surface analysis method was used to optimize the process conditions of liquefying acorn starch with thermostable α-amylase. The results showed that the DE value of 13% as the best liquefaction index, the optimal liquefaction conditions were 31 min of liquefaction time, 95 ℃ of liquefaction temperature, 6.6 of liquefying p H value, 13 U/g of thermostableα-amylase, at this point, in combination with the production practice, the value of the liquefied liquid DE was 12.91%, then through single factor experiments, the process conditions of saccharifying acorn starch liquefying liquor with pullulanase and glucoamylase were investigated, the saccharification conditions were optimized to be 25 U/g of pullulanase, 130 U/g of glucoamylase, 60 ℃ of saccharification temperature and 10 h of saccharification time, at this optimal condition, the content of ( IG2+ P + IG3) was predicted to 36.11% ± 0.17%, the optimum concentration of α-glucosidase in transglycosylation process was1.5 U/g, under the optimal condition, the total content of isomaltose, panose, and isomaltotriose were 36.27% ± 0.18%.