Oil from Schizochytrium sp. was used to separation and purification the docosahexaenoic acid （C22 ∶ 6） in two steps by high-speed countercurrent chromatography （HSCCC）. The selected liquid phases were heptane-ethyl acetate-methanol-water （15 ∶ 1 ∶ 15 ∶ 1， V/V/V/V） for the first step and heptane-methanol-water （5 ∶ 6 ∶ 1， V/V/V） for the second. The separation was followed by UV detection at 210 nm. The sample size， flow velocity， revolution speed and column temperature as the separation conditions of the high-speed countercurrent chromatography were optimized by using single factor experiments and response surface analysis. the best conditions were followed： sample size 180 mg， flow velocity 3 mL/min， revolution speed 910 r/min， column temperature 13 ℃. Under these conditions， docosahexenoic acid from Schizochytrium sp. was separated and purificated by two steps high-speed countercurrent chromatography， the docosahexenoic acid purities were 90.06% and 99.61% measured by high performance liquid chromatography（HPLC） and UV detector， the recovery ratios were 95.27% and 93.81%.