Abstract: The fructose,glucose and sucrose in tobacco were determined by high performance liquid chromatography with a High-Performance Carbohydrate cartridge column and a differential refractive index detector at isocratic mode using a premixed solution of 72% acetonitrile-28% water(v/v) as an eluent.The results showed that intra-day and inter-day precisions were in the range of 0.46%-1.54% and 0.96%-4.74%,respectively.The recovery was between 97.0%-102.3%.The limits of detection of fructose,glucose and sucrose were 95,107 and 105 μg/mL,respectively.This method meets the requirements for the routine analysis of these sugars in tobacco.