The invention discloses a method for separating beta-lactoglobulin from desalted whey powder. According to the method, desalted whey powder is used as a treatment object which is subjected to ammonium sulfate salting out, primary ultrafiltration desalting, primary ion-exchange column chromatography, secondary ultrafiltration desalting, secondary ion-exchange column chromatography, gel chromatography and final separation so as to obtain beta-lactoglobulin with relatively high purity. Compared with the prior art, the method has the advantages that firstly, the operation is convenient, the process is simplified, related requirements are lowly required, and the operation can be carried out in a small laboratory; secondly, operators are lowly required, and the popularization and the application are convenient; thirdly, beta-lactoglobulin is relatively high in separation purity, the strip is clear and the separation is thorough; fourthly, the application range is wide, and different varieties of protein can be separated.