Abstract: To establish a determination method of polysaccharides in Polygonum perfoliatum L. and the optimum polysaccharides determination conditions were investigated using the response surface methodology (RSM). The contents of total sugars and reducing sugars were measured by phenol-sulfuric acid method and 3, 5-dinitrosalicylic acid (DNS) method, respectively. These test used glucose as the standard substance. Thus the polysaccharides contents was calculated. On the basic of the results of the single factor experiments, based on a five level, three variable central composite design (CCD) -RSM, was employed to obtain the best possible combination for polysaccharides determination. The optimum determination conditions were as follows:total sugars:phenol reagent (1.7 mL), sulfuric acid (8.7 mL), reaction time (11 min);reducing sugars:DNS reagent (2.7 mL), coloration time (10 min), coloration temperature (81℃). Under these onditions, average recoveries of total sugars and reducing sugars in the test were 102.26% with RSD=4.03% (n=9) and 102.89% with RSD=2.66% (n=9), respectively. The content of polysaccharides in Polygonum perfoliatum L. was 46.72 mg/g. This method is sensitive and stable, which could be used to determine the contents of polysaccharides in Polygonum perfoliatum L. with good reproducibility.