【Objective】This study aims to develop a simple, rapid and sensitive fluorescence detection method for residual ofloxacin (OFL) in liquid samples, broaden the applications of copper nanoclusters and providing a theoretical foundation for portable diagnostic devices.【Method】A one-pot synthesis method was employed to prepare glutathione-stabilized copper nanoclusters (GSH-CuNCs) with excellent water solubility and optical properties. In an acidic condition, GSH-CuNCs and OFL have maximum emission peaks at 425 nm and 510 nm respectively upon excitation wavelength at 340 nm. With OFL, the green fluorescence intensity of the system at 510 nm was significantly enhanced compared to GSH-CuNCs, meanwhile the blue fluorescence intensity at 425 nm was slightly decreased. Thus, a GSH-CuNCs-based ratiometric fluorescent probe was constructed for the simple rapid, sensitive and selective detection of residual OFL in the enviroment.【Result】After optimization of the experimental conditions, the ratiometric fluorescent probe exhibited a linear response to OFL concentrations in the range of 0.005-10.000 μmol/L, with a detection limit (LOD) of 1.5 nmol/L. This ratiometric probe has been applied to detect OFL in the actual spiked water and milk samples. Applying with spiked real sample, the recovery rate of OFL was 93.6%-103.3%, and relative standard deviations (RSD) were all less than 10%.【Conclusion】The developed ratiometric fluorescent probe offers the advantages of simplicity, low cost, high sensitivity, and excellent selectivity, enabling rapid and accurate detection of residual ofloxacin in real-world applications.