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Tabanus bivittatus Mats., a traditional Chinese medicine, is commonly used for cardiovascular disorders treatment including atherosclerosis. There have been only a few researches on its chemical components, and no detailed report has appeared on its fatty acids. To develop a simple and effective method for the extraction of total fatty acids from Tabanus bivittatus Mats., the Soxhlet extraction (SE) condition was optimized with response surface methodology. The fatty acid composition of the extract were determined by GC-MS with previous derivatization to fatty acid methyl esters (FAMEs). The major fatty acids in Tabanus bivittatus Mats. were oleic acid, palmitic acid, linoleic acid, palmitoleic acid, and stearic acid, and the unsaturated fatty acids occupy 63.9% of the total fatty acids.