This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L-1 h-1 and 80.1 mg lactosucrose U enzyme-1 h-1, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides.