Abstract: In this study,5- HMF determination was carried out by high performance liquid chromatography in monosaccharide- acid catalytic system with direct injection.The optimization of the determination was approached using Agilent Lc1260 HPLC and UV detector.The detection wavelength was 284 nm and the mobile phase was 5%( 5 /95,v/v) methanol deionized water solution.By the analysis of measurement conditions( detector wavelength,mobile phase ratio and velocity,filter ways) and evaluation( Selectivity,accuracy,detection limit,recovery rate,etc),the standard curve equation was y = 0.006x-0.137,the correlation coefficient R2 was 0.999.The accuracy was high and the sample recovery rate was from 98% to 102%.The proposed methodology was applied to analysis of5- HMF successfully in samples. The analytical performance of the method made it suitable for the routine determination of samples.The sample solution measured out pure peaks and there were no impure peaks.