We devised an optical assay for glucose based on the genetically-engineered glucose/galactose binding protein (GGBP) from E. coli and phase-modulation fluorometry. A single cysteine mutation was introduced at position 26 of GGBP. When labeled with the sulfhydryl-reactive probe I-ANS, GGBP showed a more than 50% decrease in fluorescence intensity with increasing glucose concentration (Kd approx. 1 μM). This is consistent with the glucose-bound structure of GGBP where residue 26 becomes more exposed to the aqueous media. Since minimal lifetime changes were observed with glucose binding, a modulation sensor was devised wherein a long lifetime ruthenium metal-ligand complex (Ru) was painted on the surface of the cuvette containing ANS26-GGBP. Glucose binding resulted in changes in the relative intensities of ANS26-GGBP and Ru which were observed as dramatic changes in the modulation at a low frequency of 2.1 MHz. The modulation measured at 2.1 MHz accurately determines the glucose concentration to ± 0.2 μM.