Fatty acids in oat flour have been analyzed by using multiple reaction monitoring mode of gas chromatograph-mass spectrometer. The samples and fatty acid standards were methylated by potassium hydroxide - methanol and boron trifluoride- methanol solutions for 15 min to produce the corresponding fatty acid methyl ester. Then, the products would be separated through DB-FastFAME (90 m×0.25 mm×0.25 μm) capillary column. Multiple reaction monitoring mode (MRM) was performed under electron bombardment ionization (EI) source, and the external standard method was carried out for quantitative analysis. 40 kinds of fatty acid methyl esters could be completely separated within 38 min. A good linear relationship was achieved in the concentration range of 0.4~40 μg/mL. The correlation coefficients of standard curve were all greater than 0.999 7. The limit of quantification of 6 fatty acids such as C20:2 was 0.003 g/100 g, and the remaining 34 fatty acids was 0.000 6 g/100 g. The standard recoveries of fatty acids were 86.6%~107.3%, and the precision was between 0.9% and 5.2%. It is a method for simple operate and high selectivity, which has a good accurate, precise and sensitive. The method is suitable for the rapid and accurate determination of fatty acids in oat flour.