The synthesis of novel prebiotics has gained attention due to a greater interest in intestinal health. In this study, lactosucrose was produced by levansucrase (LS, EC 2.4.1.10) catalyzing the transfructosylation of sucrose and lactose with whey (WP) and milk permeate (MP) as lactose sources. Selected functionalized supports were investigated for the immobilization of LSs from Gluconobacter oxydans (LS1) and Vibrio natriegens (LS2) by heterofunctional attachment. The highest immobilization protein yields of 94 % and 87 % and retained activities of 55 % and 98 %, respectively, were achieved upon the immobilization of LS1 and LS2 on Relizyme^TM EP403/S functionalized with iminodiacetic acid (IDA)-cupric ions (Cu2+). The immobilization of LSs enhanced the reaction selectivity towards transfructosylation. Moreover, post-immobilization treatments (alkaline pH incubation, polyethylenimine (PEI) cross-linking) impacted the enzyme activity of the immobilized enzymes resulting in lower retained activities (11–74 %) but allowed them to achieve a higher thermal stabilization (up to 53 times increase), particularly with the high pH treated immobilized V. natriegens LS2. The identified optimal conditions for lactosucrose synthesis with high selectivity by free and immobilized Relizyme^TM EP403/IDA-Cu immobilized LS2 resulted in 117 and 101 g/L, respectively, using WP as a lactose source. Lastly, the Relizyme^TM EP403/IDA-Cu immobilized LS2 was successfully reused 3 consecutive times.