A simple and convenient method for simultaneous determination of two major effective components (oleic acid and linoleic acid) in Coix seed oil has been developed. The sample was analyzed in a RP-C18 column with acetonitrile: tetrahydrofuran: water (containing 0.4% HAC) (77:3:20, v/v/v) as a mobile phase, and the differential refractive index detector was used in HPLC. The column temperature was fixed at 30°C and the flow rate was 1.0 ml/min. The average recovery rate of oleic acid and linoleic acid are 102.83% and 99.33%, the RSD (n=6) are 1.24% and 2.27%, respectively. The standard curves of oleic acid and linoleic acid presented the favorable linear correlation over the range of 0.098-10.05 mg/ml and 0.101-10.38 mg/ml(R2 >0.999). The method reported in this paper is reliable and relatively simple with a good reproducibility for the determination of oleic acid and linoleic acid in Coix seed oil comparing with the existing methods.