Abstract: Disclosed is a method for preparation of a chitosan oligosaccharide composition capable of increasing a content of disaccharide in chitosan oligosaccharide with excellent effects of reducing HbAlc and blood glucose as well as excellent thermal stability. The method for preparation of a chitosan oligosaccharide composition includes: adding chitosan to distilled water to swell the chitosan; firstly treating the chitosan with a chitosanase to prevent the chitosan solution from becoming a state of being viscose, then, adding an organic acid to a mixed solution of the chitosan and chitosanase at a predetermined time interval bit by bit over several additions; allowing mutual reaction between the chitosan, chitosanase and organic acid; and inactivating the chitosanase to obtain the chitosan oligosaccharide composition. Further, a medicine and/or health supplement food which is effective in preventing and treating diabetes, including at least 40% of disaccharide without monosaccharide, is also disclosed.