A novel and simple fluorometry method has been developed and validated for the determination of norfloxacin (NOR) on the basis of the fluorescence quenching of thioglycolic acid (TGA)-capped CdTe/ZnS nanoparticles (NPs) by NOR. The functionalized CdTe/ZnS NPs were successfully synthesized in aqueous medium with TGA as the capping ligand. TGA-capped CdTe/ZnS NPs were characterized by using X-ray diffraction (XRD), transmission electron microscopy (TEM), particle size measurements, and Fourier transform infrared (FTIR) spectroscopy. Owing to a specific interaction, the fluorescence intensity of CdTe/ZnS NPs is clearly reduced in the presence of NOR. Under optimal conditions, the relative fluorescence intensity of CdTe/ZnS NPs is linearly proportional to NOR concentration from 0.2 to 140 μM with a detection limit of 0.082 μM. This probe was also successfully utilized to determine the NOR in pharmaceutical formations. The quenching mechanism was also investigated. The proposed method is simple, rapid, and practical.