Abstract: This study aimed to establish a time-resolved fluorescence immunoassay method for the simultaneous quantitative detection of fumonisin B₁, B₂ and B₃ in corn. The EDC/NHS active ester method was used to prepare time-resolved fluorescent microspheres and fumonisin antibody conjugates. Fumonisin antigen was immobilized on a nitrocellulose membrane as the test line. By optimizing the number of test line, a competitive immunoassay method for detecting fumonisin was eventually established. Subsequently, the sensitivity, specificity, accuracy, precision, and correlation with the national standard method were evaluated for the established method. The detection limits of the established method by using two test line pattern were 107.68~168.28 μg/kg, and the quantification limits were 283.46~444.63 μg/kg. The cross-reactivity with fumonisin B₁, fumonisin B₂ and fumonisin B₃ were 100%, 85.59% and 72.72%, respectively, and no significant cross-reactivity was observed with five other common mycotoxins. The recovery rates ranged from 88.37% to 117.42%, and the coefficients of variation were lower than 10%. The conformity of the results obtained by the established method, as compared to the immunoaffinity column purification-high performance liquid chromatography (IAC-HPLC) method specified in the national standard GB 5009.240-2016, fell within the range of 92.17% to 107.21%. The established time-resolved fluorescence immunoassay method meets the requirements for rapid on-site quantitative detection of fumonisin B₁, B₂ and B₃ in corn.