Objective: To analyze and evaluate the uncertainty of the determination of folic acid content in milk powder by microplate method. Methods: The content of folic acid in infant formula milk powder was determined by microplate method according to GB 5009.211—2022 "National Standard for Food Safety-Determination of Folic Acid in Foods". Various experimental factors affecting the test results were analyzed and calculated according to the uncertainty evaluation methods and guidelines such as JJF 1059.1—2012, CNAS-CL01-G003:2011 and CNAS-GL05:2011, and the synthetic standard uncertainty and expanded uncertainty were obtained. Results: The expanded uncertainty U₉₅=22.08 μg/100 g（k=2）, and the folate content of the sample was （282.33±22.08） μg/100 g. The uncertainty mainly comes from the process of microplate inoculation, the fitting of standard curve and the repeatability of measurement. Conclusion: The accuracy of pipettes and microbial factors have a great impact on the experimental results, so it is necessary to ensure the quality of the instrument and strictly follow the standardized procedures to minimize the uncertainty.