The diversity of functions associated with carbohydrates in physiology and pathophysiological conditions has sparked significant scientific interest in understanding their molecular interactions for enabling the development of therapeutic and diagnostic interventions. Capitalizing on the affinity of reactive blue 221 (RB221) dye toward carbohydrates, herein, we demonstrate the potential of RB221 as a new carbohydrate-recognizing moiety. For this purpose, RB221 is immobilized on the surface of polyethylenimine (PEI)-functionalized iron oxide nanoparticles (IONPs-PEI) to produce an IONPs-PEI/RB221 construct. The RB221-carbohydrate interaction is determined by exposing the IONPs-PEI/RB221 to aqueous solutions of nine different carbohydrates. The interaction with d-maltose, d-sucrose, d-lactose, d-fructose, d-glucose, l-fucose, d-arabinose, d-mannose, and d-galactose suggests that the number of molecules of these carbohydrates that adsorbed on IONPs-PEI/RB221 per RB221 molecule is approximately 2, 2, 3, 3, 3, 4, 4, and 5, respectively. Furthermore, RB221 immobilized on a tapered optical fiber surface is demonstrated for glucose sensing using phase shift fiber cavity ring-down spectroscopy (PS-CRDS). Integrating RB221-functionalized tapered optical fiber in the PS-CRDS-based sensor results in a 47-fold increase in sensitivity over bare fiber with a minimum detection limit of 2 pg/mL. This study highlights the potential of RB221-carbohydrate interactions for developing affordable and reliable sensors.