Based on CNAS-CL07： 2011， the uncertainty for biotin microbiological assay in formula refer to GB 5009.259－2016 should be evaluated. According to JJF1059.1-2012 and GB/Z22553-2010， the sources of uncertainty for the method were explored. The evaluation involves repeatability of measurement， weighing of sample and sample dilution， standard solution preparation， standard curve fitting and measured deviation of UV-Visible spectrophotometer. By analysis， the heft of uncertainty and expanded uncertainty were calculated. The content of biotin in cereal meal for infant was （32.49 ± 2.64） μg/100 g， P = 95%， k = 2. The most significant factor that contributed to the determination of biotin was microbiological factors， they were shown in standard curve fitting and repeatability of measurement.