Glucose sensing is used as a model to explore the advantages and problems deriving from the use of either enzymes or sugar binding proteins to develop stable fluorescence biosensors. We report on a novel approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apo-enzymes as non-active forms of the protein which are still able to bind the substrate/ligand. We also review studies in which derivatization of a naturally thermostable sugar-binding protein with a fluorescent probe allows quantitative monitoring of glucose binding even after immobilization on a solid support.