Abstract: OBJECTIVE An high-performance liquid chromatographic method with an evaporative light-scattering detector has been developed for the separation and determination of six kinds of fatty acids in rape bee pollen. METHODS An Agilent ZORBAX SB-C₁₈ analytical column(4.6 mm×250 mm, 5 μm) with methanol-acetic acid aqueous solution as mobile phase at a flow rate of 1.2 mL·min^-1 were applied. The column temperature was set at 25 ℃. The parameters of ELSD were as follows: the plus was 6.0; the temperature of the drift tube was 30 ℃, the carrier gas pressure was 0.3 MPa. RESULTS The separation degree of stearic, linolenic, linoleic, palm, oleic and myristic acids were above 1.5, and their recoveries ranged from 95.3% to 103.7%, the relative standard deviations(n=6) were 2.2%, 2.0%, 1.0%, 1.4%, 1.4%, 2.3%. CONCLUSION The method is simple, accurate and reproducible, it’s suitable for the simultaneous determination of the six high fatty acids in rape bee pollen.