Abstract: The presented method was developed to determine the concentration of thymol residues in honey by ultra performance liquid chromatography with a fluorescence detector. The thymol in honey was extracted by 40% acetonitrile-water solution. After centrifugation and filtration, the separation of thymol was performed on an Accucore aQ liquid chromatography column (150 mm×2.1 mm, 2.6 μm) with the gradient elution of acetonitrile and water (containing 0.1% formic acid) as mobile phases, and quantitated by external standard method with fluoresce detector (excitation wavelength 274 nm, emission wavelength 297 nm). A good linear relationship (R²=0.9999) was observed with concentration ranging from 0.01 μg/mL to 3.00 μg/mL. The average recovery of thymol was from 87.4% to 106.9%, the coefficient of variation was between 0.7% and 7.3%, and the limit of quantification was 0.10 mg/kg. The developed method was applied to determine residual thymol in 48 commercial honey samples, and the thylmol was only detected in one sample. The presented method is simple, quick, reliable, and suitable to be used for batch screening and quantitative determination of residual thymol in honey.