In this study, α-lactalbumin and β-lactoglobulin, the main ingredients in whey, were separated and purified step by step by different methods. Two-step ammonium sulfate precipitation method was used to primarily concentrate bovine whey protein, so that the protein content was increased by 4 times. The optimal parameters of PEG1500/（NH₄）₂SO₄ two aqueous phase extraction system were optimized by response surface methodology, the extracted protein fraction was further purified, so the protein concentration was further increased to 815.6 mg/g. Followed by Sephadex G-75 gel chromatography and DEAE-Sepharose Fast Flow ion-exchange column chromatogra-phy, the purity of α-lactalbumin and β-lactoglobulin reached 85%.