Isomaltooligosaccharides were prepared from puffed rice flour by a One-step processing method under different conditions, and their physicochemical properties were evaluated. Sample was prepared by allowing puffed rice flour to pass through a 50 mesh standard sieve and segregated into experimental groups (PR15-60, PR20-60, PR15-65, and PR20-65) according to the substrate concentrations (15%, 20% w/v) and reaction temperatures (60 , 65 ). Enzyme reaction of puffed rice flour and water mixture was performed for 0, 1, 6, 24, and 48 h using commercial enzymes (Maltogenic amylase, Promozyme D2 and Transglucosidase; amount: 0.5% (w/v) to obtain the corresponding isomaltooligosaccharides. The reducing sugar content, dextrose equivalent, and total soluble solids of the reactants increased with increasing reaction time. The carbohydrate composition and amount of isomaltooligosaccharides with degree of polymerization (DP) 2 to DP 7 in the rice flour were examined by HPLC with an evaporative light scattering detector. Because of the high carbohydrate composition, the PR20-65 group showed the highest isomaltooligosaccharide content after 6 h of reation (138.47 mg/mL). After 24 h of reaction, the amount of isomaltooligosaccharides (DP2-DP7) and the isomaltooligosaccharides/total carbohydrate ratio in this group were 135.00 mg/mL and 68.04%, respectively.