Abstract: A new method was developed for the determination of fatty acids in Arabidopsis seeds by ultrasonic extraction gas chromatography. The effects of experimental conditions such as ultrasonic time, heating temperature, ultrasonic power, static extraction time and type of extractants on the determination of fatty acids in Arabidopsis seeds were investigated in detail. The sample pretreatment conditions and the detection conditions of gas chromatography were optimized. The results showed that the optimal sample treatment conditions were as follows: methyl esterification ultrasonic heating temperature of 90 ℃, ultrasonic power of 400 W for 30 min, extractant of n-hexane, and centrifugation at 4 000 r/min for 5 min after the addition of extractant. Under the optimal conditions, the fatty acids had good separation effect and linearity of the standard linear curve. The limit of detection (LOD) was 2.9~21.5 mg/kg, the limit of quantitation (LOQ) was 14.5~94.0 mg/kg, and the precision was 0.5%~7.9%. The method requires less sample amount, is simple, rapid and accurate and is also suitable for the determination of fatty acids in some oil-bearing crops such as Rapeseed, Peanut and Soybean.