Abstract: In order to determine the amount of organic acids in root exudates, the authors used spectrophotometer measurement method to analyze the linear relationship between the acetic acid, malic acid and citric acid, and the orthogonal test design to determine the reaction condition of the optimal parameters and the optimal combination, thus to measure the amount of the organic acids of‘Nongda202’, NC89 and K326 in root exudates. The results showed that the optimal condition for the color reaction was as follows: 0.08 mol/L hydrochloric hydroxylamine, 0.4 mol/L DDC, 0.7 mol/L FeCl3, 50℃ , 30 min reaction time and 520 nm wavelength. The advantages of the established method included wide linear range, high accuracy, high recovery rate and low detection limit. Therefore, the colorimetric method of the ferric hydroxide, which was simple and easy to operate, was a good method to determine the total amount of organic acids in root exudates of tobacco.