Abstract: A rapid and simple method was established for simultaneous determination of three main fatty acids in chrysalis oil based on high performance liquid chromatography- evaporative light- scattering detection (HPLC-ELSD) . Chromatographic separation was carried out in gradient mode by using a Hanbon Lichrospher C18column (5μm, 250mm×4.6mm) with mobile phases of acetone-water (2% acetic acid) (v/v) . The fatty acids were in baseline separated, respectively. The calibration curves were plotted on the double logarithmic scales. The linear ranges of linolenic acid, palmitic acid and oleic acid were 0.2 ².0g/mL, 0.1 ².0mg/mL and 0.2 ~ 2.5mg/mL, respectively. R2>0.99, the limits of detection (S/N=3) were 0.05mg/mL, the recoveries were 97%~ 107% and the relative standard deviations were below 2%. The analytical method established in this work could be applied to accurate and rapid determination of three fatty acids in chrysalis oil.