© 2020 Elsevier LtdBioactive oligosaccharides are important compounds for development of functional foods and ingredients. In this study, lactose derivative hetero-oligosaccharides were produced by acceptor reaction of glucansucrase E81 with whey. The synthesis of oligosaccharides was monitored by Thin Layer Chromatography (TLC) analysis followed by 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy analysis which demonstrated the glycosylation of lactose with α-(1 → 2), α-(1 → 3) and α-(1 → 4) linkages. Liquid chromatography–mass spectrometry (LC-MS) analysis demonstrated the formation of DP 7 oligosaccharides from whey. The synthesized oligosaccharides were not digested under in vitro human gastric juice and α-amylase conditions. The prebiotic effect of lactose derivative hetero-oligosaccharides was tested and no growth in pathogenic strains was observed whereas certain probiotic strains utilised lactose derivative hetero-oligosaccharides as Carbon source and their growth was promoted. These findings can be important to reveal the potential of glucansucrases to produce functional oligosaccharides from different sources including waste materials.