Abstract: An aqueous two-phase system of PEG400/ ( NH4) 2SO4was used to extract total flavonoids from Pine Pollen. The composition of the aqueous two-phase system was 26% PEG400-20% ( NH4) 2SO4.Effects of the mass fraction of Na Cl, p H and temperature of aqueous two-phase system on the partition ratio and extraction rate of total flavonoids from Pine Pollen were studied through the single factor experiments. The optimal extraction conditions were obtained by the orthogonal experimental design.The optimum conditions were as follows: the mass fraction of Na Cl at 2%, p H at 4 and temperature at 40 ℃, and the extraction rate can reach up to 98.93%.The Na Cl addition had the most effect on flavonoids partition.The aqueous two-phase extraction has the advantages of simple and convenient procedure, moderate aqueous environment, shorter extraction time and higher extraction rate, which is effective for the separation and purification of total flavonoids.