Abstract: The reaction condition and extracting condition of the anthrone colorimetric method to determine the soluble sugar content of cotton were investigated in this paper. The results showed that the optimum determination condition was: detector wavelength of 575 nm, color- display reagent consumption of 5 mL, reaction time of 10 min, reaction stalling time of 10 min to 1 h. Under this condition, there was a good linear relationship between the concentration of glucose and absorbance (C= 0.1318A + 0.0004， R2=0.9992). The limit of detection (LOD) for glucose of the proposed method was 0.003 mg/mL. The relative standard deviation (RSD) and the average recovery rate of the method was 0.04 and 100.3% , respectively, which denoted high repeatability and precision. Hence the established method is simple, economical, rapid and accurate, and suitable for routine analysis of bulk samples.