The invention discloses a method for separating cholesteryl ester from lanolin. The method comprises the following steps of: (1) taking lanolin as a raw material, and preparing a raw material liquid from the lanolin and a hydrophobic organic solvent; carrying out fractional extraction by taking one or more than two mixed solvents from moderate polar or polar organic solvents as extracting agents and a solvent which is the same as the raw material liquid solvent as a washing agent to obtain extraction liquid; and (2) carrying out vacuum concentration, washing with water and drying to obtain the cholesteryl ester. In the method, the organic solvents which are frequently-used in the industry are adopted, and the cholesteryl ester with a higher content can be efficiently separated from the lanolin, so the method has the advantages of high separation efficiency, less solvent consumption, high productivity, low cost, safety, environmental protection, easy industrial production, and the like.