Oil from Chlorella minutissima was used to separate and purify eicxcosapentanoic acid (EPA) by using high-speed countercurrent chromatography (HSCCC) combined with evaporative light-scattering detection (ELSD). The optimal 2-phase solvent system for separation of EPA by HSCCC was acetonitrile-heptane-acetic acid-methanol (5∶4∶1∶1, V/V/V/V). The main experiments were determined and optimized by response surface methodology (RSM). The best conditions with flow-rate at 3.0 mL/min, revolution speed at 913 r/min, and column temperature at 21 °C were obtained. Under these conditions, 75 mg EPA with a purity of 99.44% was obtained from 500 mg oil by HSCCC, as detected by HPLC combined with ELSD.