Here a useful gas chromatography mass spectrometry method for determining levels of fatty acids in the maternal serum was developed and validated. Forty-five mixed standards of fatty acids methyl ester of more geometrical isomers and biological significance were separated on a capillary column with higher polarity and thinner film thickness. The total run time was approximate 36 min. Twenty serum fatty acids methylated by a H2SO4-CH3OH-toluene mixture were subsequently identified. Measurements for each fatty acids methyl ester were linear over a wide range (0.05–100μg/mL, correlation coefficient > 0.99).The limits of detection and quantification for the targeted fatty acids were <9 and 22 ng/mL, respectively, satisfactory recoveries occurred in 75.07–98.09% of cases, and the relative standard deviation for each fatty acids was <12%. On a conditional logistic regression model, a high level of eicosapentaenoic acid was a protective factor against a low development quotient. This method was successfully applied to evaluate the association between maternal fatty acids level in early pregnancy and mental retardation in 2-year-old children.