The work aims to design a method for determining the starch content in tipping paper for cigarette. Glucoamylase was used to degrade the starch in the tipping paper for cigarette. The glucose of the enzyme hydrolysis product in the solution was measured by 3,5-dinitrosalicylic acid colorimetry (DNS colorimetry) and the starch content in tipping paper for cigarette was calculated. Based on the results of the single factor experiments, the response surface methodology was used to optimize the optimal conditions of starch content determination in tipping paper for cigarette with enzyme hydrolysis and establish the mathematical model. Meanwhile, the precision and recovery rate of the method were tested. The optimal determination conditions of starch in tipping paper for cigarette were glucoamylase dosage of 2.64 mg・mL–1, reaction time of 35 min, reaction temperature of 53 ℃, correlation coefficient of 0.999, detection limit of starch of 1.1 mg・L–1, relative standard deviation of 1.02% and recovery rate of 95.2%-105% respectively. The method is easy to operate, with high accuracy and it can be used to determine the starch content in tipping paper for cigarette.