An uncertainty evaluation method for the microbiological determination of biotin in infant formula was developed in order to control the quality of test results and improve the detection accuracy, a method for uncertainty evaluation of milk powder by method was established. According to the requirements of the Evaluation and Expression of Uncertainty in Measurement (JJF 1059.1—2012) and the Guide to the Evaluation and Expression of Uncertainty in Measurement (GB/T 27418—2017), the uncertainty components were estimated for the determination of biotin in infant formula by the method specified in the National Food Safety Standard Determination of Biotin in Foods (GB 5009.259—2016). It was found that the greatest uncertainty resulted from standard curve fitting and repeatability test. The uncertainty for the biotin content of three infant formula samples was as follows: sample A (19.87 ± 2.84) μg/100 g, B (30.35 ± 4.41) μg/100 g, and C (53.15 ± 7.15) μg/100 g (in the 95% confidence interval, coverage factor (k) = 2). As the content of biotin in samples increased, the expanded uncertainty also increased.