Separation of EPA and DHA from fish oil fatty acid ethyl ester (FAFE) by urea adductive crystallization method was carried out in the supercritical carbon dioxide (SC CO2) as a solvent. Our results showed that SC CO2 is a good candidate as a solvent in the urea adductive crystallization to separate FAFE by the number of unsaturated bonds. Compared to the separation process using methanol. SC CO2 yielded better performance in the overall selectivity of EPA and DHA. The effect of process variables on separation of EPA and DHA was discussed in detailed. A hybrid technology of SC CO2 fractionation and urea adductive crystallization with SC CO2 was conformed as a viable process to separate and concentrate EPA and DHA from fish oil.