Abstract: To investigate the effect of derivatization with sulfuric acid and methanol on measuring the mass fraction of levulinic acid in tobacco, the influences of the derivation time and temperature were analyzed. In addition, five sugars were investigated under the conditions of derivatization on possible formation of levulinic acid methyl ester. Relationships between the mass fractions of fructose and sucrose and the mass fraction of levulinic acid in 263 tobacco samples were analyzed and compared. The results showed that: 1) The effects of the derivatization time and temperature on the response of levulinic acid methyl ester were different from the response of other organic acids methyl ester in methyl esterified tobacco sample, and those differences were related to the tobacco matrix. 2) Fructose and sucrose could produce levulinic acid methyl ester under the conditions of derivatization, while glucose, maltose and inositol could not. 3) The levulinic acid methyl ester mass produced by sucrose under the conditions of derivatization was only half of that by fructose at the same mass. 4) The mass fraction of levulinic acid in tobacco detected by the proposed derivatization method with sulfuric acid and methanol was affected by the fructose and sucrose levels in tobacco.