The invention provides a method for extracting and separating rufuslactone from a Lactarius rufus entity. The method comprises the following steps of: (1) crushing the Lactarius rufus entity after the Lactarius rufus entity is dried, dipping and extracting the crushed Lactarius rufus with methanol at room temperature to produce a methanol extracting solution; (2) distributing and extracting the methanol extracting solution by a chloroform-water system and reclaiming chloroform to produce an extraction liquid, transferring the extraction liquid into a silica gel column and eluting the silica gel column in gradient by a petroleum ether-acetone system to collect an eluent; (3) transferring the eluent acquired in step (2) into a gel column to elute the gel column by a methanol-chloroform system, detect the eluent with thin layer chromatography and merge main compositions in the eluent to produce a rufuslactone pure product. The method has the advantages of short extraction time, simple and easy operation of a raw material separation process, high yield, accessible mass production and so on.