Abstract: The three phase extraction method can avoid the alcohol precipitation process of polysaccharides to separate polysaccharides in a short time. At the same time,the protein and flavonoid in the aqueous extract of ionic water can also be segregated in a certain extent. In this paper,Lycium barbarum polysaccharides were separated by this method,and the three phase extraction properties of tertiary butanol-(NH₄)₂SO₄-aqueous extract system were investigated. The effects of four factors,including the amount of(NH₄)₂SO₄ in the extract,the amount of tertiary butanol,pH and temperature of the extract,on the extraction rate of polysaccharides,protein and flavonoids from Lycium barbarum extract were studied,and the extraction conditions of polysaccharides were optimized by orthogonal experiment. The optimum extraction conditions were as follows:10 mL extract(NH₄)₂SO₄ was added to 30%(w/v),tertiary butanol was added to 10 mL,the temperature was 35 ℃ and pH was 6,the extraction rate of polysaccharides reached 95.31%,the protein extraction rate reached 83.45%,and the extraction rate of flavonoids reached 50.66%. The infrared spectra and antioxidant activities of polysaccharides showed that there was no significant difference in the chemical structure and antioxidant activity of polysaccharides obtained by three phase extraction and ethanol precipitation. Therefore,the system would be a feasible and efficient method for separating and purifying Lycium barbarum polysaccharides.