To evaluate the measurement uncertainty in the microbiological determination of the vitamin B₆ content in milk powder, we estimated various uncertainty sources according to the National food safety standard for the determination of vitamin B₆ in foods (GB 5009.154-2023) and the Evaluation and expression of uncertainty in measurement (JJF 1059.1-2012), and calculated combined standard and extended uncertainties. The results showed that the content of vitamin B₆ in milk powder was (9.34 ± 1.23) mg/kg (k = 2, p = 95%). Through uncertainty component analysis, it was found that measurement repeatability and the preparation of standard solution had a significant impact on the determination of the vitamin B₆ content in milk powder.