The reason of the influence of acid hydrolysis on total sugar determination in sweet sorghum by 3,5-dinitrosalicylicacid method was analyzed. Fructose could completely degrade when the ratio of 6mol/L hydrochloric acid to sugar liquid was 2∶1 in a boiling water bath for 30min. Under such conditions, the measured values of glucose, fructose and sucrose by DNS method were 100%, 2.5% and 52.2% of the corresponding theoretical values. Meanwhile, the fructose solution after hydrolysis appeared a pseudo peak of glucose in the HPLC chromatogram, which caused glucose value higher than the real value. The determination of fructose and sucrose was impacted by the acid concentration, hydrolysis time and hydrolysis temperature. Fructose degradation during the acid hydrolysis caused the measurement error in the total sugar determination in sweet sorghum by DNS method. When determinate total sugar content in raw material contained fructose and sucrose by DNS method, it should ensure the complete hydrolysis of sucrose, and no degradation of fructose, otherwise it would result in inaccurate measurement.