Abstract: Using 1.0 g/L 6-hydroxy-2,4,5-traminopyrimidine as the fluorescence derivative,the derivatives with strong fluorescence were generated at 40 ℃ for 30 min under the condition of reaction with vitamin C in aqueous solution at a pH of 6.5±0.5.The extraction,derivation and chromatographic conditions of vitamin C were studied and optimized. Under the condition of using pH=4.0 ammonium acetate buffer(v)+ acetonitrile(v)+ methanol(v)=80+15+5 as the mobile phase,the derivative had stable chromatographic behavior and generates a strong fluorescence signal. Its content was detected by high performance liquid chromatography-fluorescence detector. Vitamin C with accurate result,the method recovery was 90.9%~96.7%,experimental data RSD≤2% or less stable,high sensitivity method detection limit of 0.2 μg/g,detection of linear range 0.05~500 μg/g,good correlation coefficient,y=93322x-1837.8,R²=0.9999,experimental operation was simple,did’t need to repeat the preparation of blank sample operation and other characteristics,was more suitable for laboratory batch testing tasks. Some references were provided for the use of 6-hydroxy-2,4,5-traminopyrimidine as a fluorescence derivative.