Abstract: To isolate β-lactoglobulin(β-Lg) environmental friendly, high efficiently and under mild conditions from whey, a novel method including selective protease treatment of whey and ultrafiltration of hydrolysates was investigated. Whey hydrolysates hydrolyzed by pepsin, trypsin, papain, and neutrase were analyzed respectively in terms of selectivity of proteases by SDS-Polyacrylamide Gel Electrophoresis(SDS-PAGE). The ultrafiltration (UF) was performed using polysulfone membrane PS-10 or polyether sulfone membrane PES-10 with 10000 molecular weight cut-off limit, respectively. The SDS-PAGE indicates that β-Lg is resistant to pepsin digestion, while α-Lactoalbumin (α-La) is not sensitive to papain. But both proteins are susceptible to neutrase. When 8% whey protein solution was hydrolyzed by 0.3%(m/m) pepsin at 37℃, with pH value of 2.1, for 2 h, all the α-La was digested and little β-Lg was degraded. The 94.6% purity and 75.6% yield of β-Lg could be reached, when ultra-filtration was performed with PS-10 membrane at 40℃, under 0.22 MPa and with pH value of 6.0. Hence, proteolysis of whey coupled with ultrafiltration treatment is feasible to separate β-Lg.