The present invention relates to a kind of method that EPA and DHA are kept completely separate from fish oil, including：By one shake well of dicyandiamide solution, split-phase, the upper and lower two-phase of separate collection are stood；Fish oil extract is dissolved in upper phase, more than mutually for stationary phase, lower phase is mobile phase, is detached using high speed adverse current chromatogram, removes mobile phase, obtains the mixture of EPA and DHA；By two shake well of dicyandiamide solution, split-phase, the upper and lower two-phase of separate collection are stood；Mixture is dissolved in upper phase, more than mutually for stationary phase, lower phase is mobile phase, is detached using high speed adverse current chromatogram, removes mobile phase, respectively obtains EPA and DHA products.The all recyclable recycling of reagent used by two dicyandiamide solutions of the present invention, theoretically EPA and DHA can 100% recycling.
