One-pot cascade reaction systems offer several process and technological benefits, completed by the opportunity of the second enzyme to act on a convenient concentration of its substrate. Most of the reported cascade reactions involve immobilization of both biocatalysts on a single support, but in this case the catalytic performance of the whole system is affected if the activity of either enzyme drops. In this work, tailor-made immobilization of two β-galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and a glucose oxidase (from Aspergillus niger) was investigated, to subsequent utilization of the best performing immobilized biocatalyst of each type for the one-pot conversion of lactose to gluconic acid and galacto-oligosaccharides. The immobilization was performed by covalent binding onto amino-terminated Ni0.4Co0.2Zn0.4Fe2O4 and Ni0,5Zn0,5Fe2O4 magnetic particles and commercial amino- and epoxy-functionalized methacrylate supports. The highest activity for β-galactosidases was achieved on Ni0.5Zn0.5-Fe2O4 magnetic particles, meanwhile the glucose oxidase showed higher affinity for the supports with epoxy active groups. The immobilized biocatalysts have been characterized in detail, demonstrating enhanced storage, pH, and thermal stability as well as better operational stability compared to the soluble enzymes.