Provided is a method of preparing a high-purity galactooligosaccharide composition by fermentation of a low-purity galactooligosaccharide mixture with a yeast strain, Kluyveromyces lactis ATCC 8585. The high-purity galactooligosaccharide composition includes at least 99% of galactooligosaccharides selected from the group consisting of galactotriose, galactotetrose, galactooligosaccharides with five or more sugar units, and combinations thereof and lower than 1% of monosaccharides and disaccharides. Also provided is a high-purity galactooligosaccharide composition and applications thereof in regulating blood glucose level and improving gut microbiota. For example, the high-purity galactooligosaccharide composition may be used to manufacture food products, beverages, health food products, nutritional supplements, and pharmaceutical compositions for patients or pets afflicted with diabetes mellitus or lactose intolerance.
