Abstract: High-speed countercurrent chromatography ( HSCCC) is a kind of liquid-liquid partition chromatography, which has the unique features of high efficiency and continuous processing capability. It has been widely used in the separation and purification of Chinese heabal medicines, the natural products and food, as well as in biochemistry and in environmental chemistry analysis. In this paper, the principle and characterization of high-speed countercurrent chromatography and its application in separation and purification of fatty acids and corresponding esters from microalgae oil, fish oil and other sources were summarized, and the application future in separation and purification of fatty acids by high-speed countercurrent chromatography were predicted.