Abstract: EPA and DHA in fish oil were quantitatively analyzed by gas chromatography(GC),and the analysis conditions for rapid detection of EPA and DHA were explored. The content of EPA and DHA in fish oil from stomach of spanish mackerel fish by ultrasound enhanced subcritical water,ultrasound assisted organic solvent,enzyme hydrolysis and supercritical CO₂ extraction were determined using the rapid GC analysis method. The results showed the peak time of EPA was 10.015 min,and DHA was 16.500 min. The heating program and shunt ratio could significantly affect the separation degree of the target peak(EPA,DHA)and its adjacent peak R₁ and R₂(p<0.01). Compared with the chromatography analysis method of fatty acids recommended in GB5009.168-2016,the method had advantages of strong pertinence(fast detection of EPA and DHA),high accuracy,good reproducibility and high efficiency. The analysis time was shortened from 82 min to 18 min. Under the best analysis conditions,the content of EPA in fish oil were 54.67,35.23,40.13 and 33.40 mg/g,respectively,DHA were 134.01,77.50,102.30 and 67.31 mg/g,respectively.