In the present study, biomolecule-stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of pazufloxacin mesilate (PZFX) for the first time. The linear decrease in the fluorescence intensity of Au nanoclusters induced by PZFX allowed for the quantitative detection of PZFX in the range of 0.15 μg/mL to 1 mg/mL, and the detection limit for PZFX was 0.2 μg/mL. Circular dichroism spectroscopy and fluorescence decay studies were then performed to discuss the quenching mechanism. In addition, practical application of the present approach was also demonstrated for real samples, which suggested its great potential for accurate analysis of similar drugs.