Abstract: OBJECTIVE To establish the method of extraction, refinement and purification for fish oil from ray fish liver, and to develop a analytical method for the determination of the content of EPA and DHA. METHODS Highly purfied unsaturated fatty acids were purified by N-hexane ultrasound extraction, H₃PO₄ degumming, NaOH depickling, active carbon decolor, rotary evaporation deodorization, salt fractionation extraction total fatty acids, urea complexation. The content of EPA and DHA was detected by HPLC. RESULTS The results showed that refined fish oil was yellow, transparent with light traing flavour and each index reached the requirement of SC/T3502-2000. The total mass fraction of refined fish oil in liver was up to 16.1%, and the content of EPA and DHA was 19.54% and 57.62%, respectively. CONCLUSION This is a feasible method for extraction, refinement and purification of fish oil in the liver of the ray. The method for detection is reliability.