Gas charomatography-flame ionization detector（GC-FID） was used for simultaneous determination of seven fatty acids in pharmaceutical rubber products. The butyl rubber stoppers were cut into small pieces, by using the buffer solution of pH 2.5, buffer solution of pH 12.0 and ethanol solution of volume fraction of 50% as extraction solution respectively, they were put in 60 ℃ oven for 2.5 h. The extraction solution was treated with boron trifluoride methanol solution as methyl esterification reagent, sealed reaction for 1 h at 60 ℃, then taking methyl heptadecate as internal standard for quantitative analysis, it was analyzed by GC-FID method. The results showed that there was a good linear relationship between peak area of methyl esterification and mass concentration of seven fatty acids in the mass concentration range of 5.0 to 100.0 μg/mL with correlation coefficient greater than 0.999, and the detection of limit was 1.0 μg/mL. The mean recoveries of seven fatty acids from spiked butyl rubber stoppers at low, medium and high concentration levels were ranged from 97.0% to 100.6% with relative standard deviation of 0.02%~2.1%. This GC-FID method is simple, sensitive and accurate, which can provide technical support for the detection and analysis of fatty acid in pharmaceutical rubber products.