Abstract: The seed oil of Microula sikkimensis Hemsl. was extracted by supercritical carbon dioxide extraction, microwave-assisted reflux extraction, ultrasound-assisted extraction and solvent reflux extraction. The experimental parameters of supercritical carbon dioxide extraction which is classic in these extraction techniques including pressure, temperature, particle size and extraction time were optimized. A simple and sensitive method for the simultaneous determination of 39 kinds of saturated and unsaturated fatty acids in seed oil using high performance liquid chromatography (HPLC) with fluorescence detection following pre-column derivatization with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) has been developed. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column in conjunction with a gradient elution. Quantitative linear range of 39 fatty acids was 0.014–14.29 µ mol/L, and excellent linear responses were observed with correlation coefficients more than 0.9992. Detection limits were in the range of 3.24–36.97 fmol (10µL, S/N 3:1). Stability of derivatives, method repeatability and recovery were evaluated and the results were excellent for efficient HPLC analysis. Fatty acids in M. sikkimensis Hemsl. seed oil with or without saponification by different extraction methods were analyzed and compared. The facile TSPP derivatization coupled with HPLC fluorescence detection allowed for the quantitation of short-and long-chain fatty acids from biological and natural environmental samples.