Isomaltooligosaccharide (IMO) is a promising dietary component with prebiotic effect, and the long-chain IMOs are preferred to short chain ones owing to the longer persistence in the colon. To establish the optimal process for synthesis of long-chain IMOs, we systematically examined the reaction condition of dextransucrase of Leuconostoc mesenteroides B-512F by changing the ratio of sucrose to maltose (varying as 1:4, 1:2, 1:1, and 2:1) and amount of each sugar (from 2% to 20%). As a result, a ratio of 2:1 (sucrose to maltose, 10:5% or 20:10%, w/v) was determined as an optimal condition for long-chain IMO synthesis (DP3-DP9) with relatively higher yields (70-90%, respectively).