Chitosan-coated CdTe quantum dots can reduce QDs toxicity and enhance its stability in aqueous solution. Chitosan-coated CdTe QDs were used as fluorescence probes to determine Gemifloxacin on the basis of fluorescence quenching method. The results indicated that the relative fluorescence intensity was linearly proportional to the Gemifloxacin concentration in the range of 3.46×10－9～3.46×10－7 g·L-1 with a linear fitting equation of F0/F=1.0637+0.016 7c(g·L-1) and the RSD was 2.7%. On the basis of fluorescence quenching method theory, it was concluded that the interaction between QDs and Gemifloxacin was a kind of static quenching through hydrogen bonding and Van der Waals force, and the binding sites value was 0.8. This method with high sensitivity and broad linear range provided a new approach to determining Gemifloxacin.