It is well known that one of the main problems in galactooligosaccharide production (GOS) via tranglycosylation of lactose is the presence of monosaccharides that contribute to increasing the glycaemic index, as is the case of glucose. In this work, as well as studying the effect of ultrasound (US) on glucose oxidase (Gox) activation during gluconic acid (GA) production, we have carried out an investigation into the selective oxidation of glucose to gluconic acid in multienzymatic reactions (β-galactosidase (β-gal) and Gox) assisted by power US using different sources of lactose as substrate (lactose solution, whey permeate, cheese whey). In terms of the influence of matrix on GOS and GA production, lactose solution gave the best results, followed by cheese whey and whey permeate, salt composition being the most influential factor. The highest yields of GOS production with the lowest glucose concentration and highest GA production were obtained with lactose solution in multienzymatic systems in the presence of ultrasound (30% amplitude) when Gox was added after 1 h of treatment with β-gal. This work demonstrates the ability of US to enhance efficiently the obtainment of prebiotic mixtures of low glycaemic index.