Abstract: In this work, the effect of the composition of polyethylene glycol(PEG) -ammonium sulfate aqueous two-phase system on the extraction rate of ampelopsin from Ampelopsis grossedentata was studied. The effects of the molecular weight of PEG, the mass fraction of PEG, the mass fraction of ammonium sulfate, pH and the mass fraction of NaCl on the extraction rate were studied by single factor experiment. Secondly, three factors with significant influence on extraction rate were selected through Plackett-Burman design, namely, mass fraction of PEG, mass fraction of ammonium sulfate and pH. Response surface methodology was used to further optimize the influential factors. The optimum extraction conditions were: PEG1000 mass fraction 28%, ammonium sulfate mass fraction 11.2%, pH6.0. Under this condition, the theoretical extraction rate was 98.82%, and the actual extraction rate was 98.53% after process verification, which was close to the predicted value and 17.53% higher than the extraction rate before optimization(71%), which was conducive to the further separation, purification and application of ampelopsin.