Multi-layered silica and titania thin films were prepared via an aqueous sol-gel route. The titania layers could efficiently entrap anti-HRP antibodies and showed increase in stability upon ageing, up to 2 months. Specific activity of the entrapped antibodies could be monitored in a reliable manner over one week. The silica films showed lower performances, as a probable consequence of large pore size. Increase in the titania precursor (TiBALDH) concentration and addition of glycerol allowed the design of tri-enzymatic multi-layered systems for the detection of lactose. The multilayer approach showed higher robustness than the co-immobilization of the enzymes in a single layer. This process is simple, solvent-free and flexible, opening the route to multi-biofunctional materials at the nanoscale.