The invention discloses a separation and preparation method of ligustilide. The method comprises the following steps by the technology of high-speed countercurrent chromatography. The method comprises the following steps: extracting ligusticum wallichii by a solution to obtain a ligusticum wallichii extract; extracting the ligusticum wallichii extract by ethyl acetate to obtain an ethyl acetate part; separating the ethyl acetate parts by high-speed countercurrent chromatography at the same time to obtain a plurality of ligustilide compounds which are respectively senkyunolide N, senkyunolide R, senkyunolide S and senkyunolide I through structural identification, wherein the purity is over 98%. The separation and preparation method of ligustilide disclosed by the invention has the advantages of simplicity in operation, few use level of solvent, no sample adsorption, loss and pollution, high efficiency and the like.