Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate Such fluorescent sensor proteins lack. generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.