A novel fluorescent derivative of glucose was synthesized by reacting d-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (γ_ex = 475 nm, γ_Em = 550 nm). The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by ¹H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasm of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway.