Abstract: Objective: To establish an analytical method for the determination of 37 kinds of fatty acid in milk powder by gas chromatography-hydrogen flame ionization detector (GC-FID). Methods: Infant milk powder samples were methylated by acetyl chloride-methanol method (10% acetyl chloride methanol solution in water bath at 80 ℃±1 ℃ for 2 h). The supernatant was collected by centrifugation, passed through a 0.22 μm organic filter membrane, and separated through SP-2560 (100 m×250 μm×0.2 μm). Then, the detection was operated by gas chromatography, and the external standard method was used for quantification. Results: The linearity of 37 kinds of fatty acid methyl esters was good in the concentration range of 0.002~0.2 mg/mL, and the correlation coefficients were all greater than 0.999. The recovery rate fell in 85.5%~100.0% for the low gradient, 91.2%~104.2% for the medium gradient, and 86.8%~103.8% for the high gradient. The precision was between 0.88% and 3.81%, and the actual sample determination results met the relevant standards. Conclusion: This method is a more rapid and better separation degree of fatty acid detection technology established on the basis of national standard, which has good practical value. The method has good stability, low cost, good reproducibility and high accuracy, and it is suitable for the determination of 37 kinds of fatty acid in infant milk powder.