[Objective]This article discussed the quantification and evaluation of the uncertainty of ELISA detection method for aflatoxin M₁ residue in milk powder.[Method]By established a mathematical models,the factors of the introduction of uncertainty are analyzed by combining the fishbone diagram,and the uncertainty introduced by them was specifically quantified and compared.Thus,at a confidence level of 95% and containing factor k=2,the extended uncertainty of the determination of aflatoxin M₁ in milk powder is：0.023 μg/kg.[Result] The M₁ content of Aspergillus flavus in milk powder determined by ELISA method in this article should be expressed as：0.480±0.023 μg/kg.Among them,the factors that have a large influence on this uncertainty, the uncertainty introduced by standard curve fitting.[Conclusion]In the future inspection,the number of additional standard samples or quality control sample tests can be increased as appropriate to reduce the impact of the uncertainty introduced by this item on the inspection results.