The invention discloses a method of separating caulerpin in pinnate caulerpa by high-speed countercurrent chromatography. The method comprises the following steps: (1) crushing pinnate caulerpa and extracting by a microwave assistant extraction process to obtain crude extract; (2) selecting a solvent system for high-speed countercurrent chromatography; (3) concentrating the crude extract to paste, dissolving the paste with a lower phase of the high-speed countercurrent chromatography solvent system to obtain high-speed countercurrent chromatography separating sample liquor; (4) collecting each flow component by adopting the high-speed countercurrent chromatography separating sample liquor; (5) adopting efficient liquid-phase chromatographic analysis on each flow component and a caulerpin standard substance, and collecting the flow component with retention time consistent with that of the standard substance through the time retention of the efficient liquid-phase chromatography to obtain a caulerpin product. According to the method, MAE (microwave assistant extraction) and HSCCC (high-speed countercurrent chromatography) are combined to realize separating and purifying caulerpin in pinnate caulerpa, so that the precision and the repeatability are improved relative to a conventional separating and purifying method. Moreover, the method is simple to operate, short in time consumption, wide in scope of application, small in environmental pollution, and suitable for industrial popularization.