Abstract: The fatty acids of walnut kernel and fermented milk were analyzed by gas chromatography with Flame Ionization Detector ( FID) .The methyl ester reaction conditions were investigated using single factor and response surface methodology based on Box- Benhnken centre design principles. The derivatization reagent addition amount, ultrasound time and ultrasonic water bath temperature were used as affected factors while the total peak area of chromatogram as response value in this study.The results indicated that the optimum conditions for methyl esterification of fatty acids from walnut kernel and walnut milk were obtained as follows: the amount of KOH-methanol 647μL, ultrasound time 5min and ultrasonic water bath temperature 43℃. Under the above conditions, 5 kinds of fatty acids were detected from 9 different walnut kernels, whose contents from high to low in an order of linoleic acid, oleic acid, linolenic acid, palmitic acid, stearic acid.The content of overall change of fatty acids, which was from walnut milk fermented by 6 kinds of lactic acid bacteria, respectively was not significant.