A polyacyl maltooligosaccharide derivative which is a precursor of a maltooligosaccharide derivative having modified groups at the 6-position or 4- and 6-positions of non-reducing end glucose unit and being effectively used as a substrate for measuring .alpha.-amylase activity, can be produced under mild conditions by crosslinking OH groups in the non-reducing end glucose unit of oligosaccharide with a benzylidene group having an alkoxy group, followed by acylation and treatment with a dilute acid.