Highly purified derivatives of maltooligosaccharides can be produced at a high yield by reacting, in a mixture of a hydrophilic organic solvent and water, a mixture of maltooligosaccharides or a substance capable of being converted into the maltooligosaccharides upon reaction with an amylase, and an o-glucosyl derivative, with the amylase.The resulting derivatives of maltooligosaccharides are useful as a substrate for the determination of .alpha.-amylase activity in a humor, physiologically active substances, natural dieteic sweetenings, coloring agents and the like.