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Fatty acids in Herpetospermum seed oil from supercritical CO2 extraction were analyzed by HPLC fluorescence detection (HPLC-FLD) with pre-column derivatization and GC-MS. After derivatizing 39 kinds of saturated and unsaturated fatty acids used 1-[ 2- ( p-toluenesulfonate ) ethyl]-2-phenylimidazole [ 4,5-f] 9,10-phenanthrene ( TSPP ) as pre-column derivatization reagent. All the fatty acid derivatives were separated with a good baseline resolution in conjunction with a gradient elution. The external standard method for the simultaneous quantitative determination of 39 fatty acids was developed and applied for the determination of the free fatty acid contents in Herpetospermum seed oil samples obtained from supercritical CO2 extraction coupled with orthogonal tests, ultrasound-assisted extraction and microwave-assisted reflux extraction. The mass percent of oleic acid, linoleic acid and linolenic acid and the ratio of unsaturated fatty acids and all fatty acids in 9 orthogonal test samples were contrasted. The results indicated that the mass percent of oleic acid, linoleic acid and linolenic acid in Herpetospermum seed oil are up to 34.65% ( 147. 14 mg/g), 22.85% (97.03 mg/g), 20. 86% ( 88. 56 rag/g), respectively, and ratio of unsaturated fatty acids and all fatty acids is 79%. Furthermore, by GC-MS method, the acid catalysis and alkaline catalysis of the methyl esterifying reaction were discussed for the analysis fatty acids in Herpetospermum seed oil, and the optimum GC-MS conditions were obtained. Simultaneously, the characteristics of HPLC and GC-MS methods were discussed about analyzing fatty acids.