A novel method was proposed for the determination of fructose and glucose content during acetic acid fermentation of Zhenjiang aromatic vinegar by high performance liquid chromatographic.Vinegar samples were pretreated with a C18 solid-phase extraction column.The chromatographic separation was performed on a Cosmosil sugar-D column at temperature of 35℃.The mobile phase,a mixture of acetonitrile-water(78∶ 22,v/v),was delivered at a flow of 1.0 mL/min.The analytes were detected using RI(Refractive index) detector and quantified by external standard method.The results showed that the linear ranges were wide,and the average recoveries ranged from 96.28% to 96.69%.This method is rapid,accurate,and suitable for the determination of fructose and glucose content during vinegar fermentation.