Silicate nanoparticles with immobilized FRET-based biosensors were developed for the detection of glucose and maltose. Immobilization of the protein biosensor in the nanoparticle was achieved through specific interaction between the hexa-histidine tag of the protein and a calcium–silicate complex of the silica matrix. Encapsulation of the biosensors preserved the affinity for the respective sugar. Compared to the free biosensors, encapsulation had a stabilizing effect on the biosensor towards chemical and thermal denaturation. The demonstrated immobilization strategy for specific sensing proteins paves the way towards the development of protein-inorganic nanostructures for application in metabolite analyses.