A novel assay of Norfloxacin with a sensitivity at the microgram level is proposed based on the measurement of enhanced fluorescence intensity signals by the interaction of functionalized nano-CdS with Norfloxacin. The CdS nanoparticles were synthesized by thioacetamide (TAA) and cadmium nitrate (Cd(NO3)2) in the alkaline solution, and the nanoparticles proved to be stable in the aqueous solution. At pH 7.4, the fluorescence signals of functionalized nano-CdS were greatly enhanced by Norfloxacin in the region of 300-700 nm characterized by the peak around 495 nm, and the surface defect-related emission located at 565 nm, However, the surface defect-related emission was unconspicuous, thus we concluded that the functionalized nano-CdS QDs (Quantum Dots) possessed excellent luminescence capability and favourable structure. At the same time, the absorption spectra and transmission electron microscopy (TEM) also proved this deduction. The external reaction conditions (such as effect of buffer system, pH, ionic strength, reaction time and temperature, colloid concentration) were discussed. The result showed that better fluorescence signals could be obtained in the condition of 0.10 mol·L-1 Tris-HCl, pH 7.4, 0.1 mol·L-1 NaCl and the reaction time 5 min. The fluorescence emission spectra of CdS QDs with increasing Norfloxacin concentration were recorded under the optimal condition. From the fluorescence intensity and peak position of nano-CdS colloidal as different concentration of Norfloxacin was added, the possible mechanism of reaction between mercapto-acetic acid capped CdS and Norfloxacin was discussed. Linear relationship can be established between the enhanced fluorescence intensity and Norfloxacin concentration in the range of 1.25-11.25 μg·mL-1 (3.92-35.27 μmol·L-1) or 11.25-100.0 μg·mL-1 (35.27-313.5 μmol·L-1).The limit of detection is 1.5×10-3 μg·mL-1, which can be applied to the determination of blood serum samples. Based on this, a new direct quantitative determination method for Norfloxacin in synthetic samples without separation of foreign substances is established. At the same time, the possible enhancing mechanism is due to the formation of exciplex during reaction between nano-CdS and Norfloxacin, providing a guidance for the study of pharmacokinetics.