Abstract A galacto-oligosaccharide product (VITAGOS™) was prepared by glycoside hydrolases (β-galactosidase from Aspergillus oryzae and Kluyveromyces lactis ) using lactose as substrate. Monosaccharide composition analysis indicated that VITAGOS™ contained 40.3% (w/w, dry basis) glucose and 51.1% (w/w, dry basis) galactose. According to high performance size exclusion chromatography (HPSEC) analysis, VITAGOS™ was a sugar mixture with degree of polymerisation (DP) ranging from 1 to 7, with DP2 and DP3 as the dominant fractions (32% and 33%, w/w). In addition, more than three fractions were observed for each DP pool indicated by the oligosaccharides profiles obtained from high performance anion exchange chromatography (HPAEC) analysis. Six sub-fractions (F1-F6, corresponding to DP1 to DP6 rich fraction, respectively) were then collected using biogel P-2 column, followed by structural characterisation through methylation analysis combined with 1D & 2D NMR spectroscopy. The detailed molecular structures of VITAGOS™ with DP up to 4 are provided.