As an optional ingredient commonly added to infant formula,arachidonic acid （ARA） is also a necessary item in the food safety test of infant formula. To analyze the content uncertainty of ARA in infant formula by gas chromatography,the uncertainty components was assessed through the determination of the process of repeated measurement,the preparation of sample,the preparation of standard solutions and testing instruments according to regulation of Evaluation and Expression of Uncertainty in Measurement. The synthetic uncertainty was 2.73%,and the expanded uncertainty was 5.46%. The content of ARA in infant formula was （0.264±0.007）g （k=2）. The results showed that the uncertainty of the measurement results of ARA content in infant formula mainly came from the repeated determination of sample （2.68% uncertainty of component）, the influence of standard solution （0.40% uncertainty of component） and the sample treatment （0.31% uncertainty of component）,while the influence of gas chromatography instrument （0.00002% uncertainty of component）was the least. In the detection process, he uncertainty brought by the manual operation of the experimenter has the greatest impact on the accuracy of the detection results,and second focus is on standard solution preparation and sample handling.