The enzyme-linked immunosorbent assay (ELISA) is used to quantitatively detect Aspergillus flavus in feed by utilizing the principle of competitive enzyme-linked immunosorbent assay (ELISA). Shake and extract Aspergillus flavus from the crushed sample with methanol/water, filter the aqueous extract, and then perform immunological testing. Add the enzyme label of Aspergillus flavus to the test well that has been added with standard or sample. After the antibody is added, the reaction begins, and the sample and enzyme label compete to bind to the antibody attached to the micropore. After 10 minutes of cultivation, pour out the solution from the well and wash away the unbound Aspergillus flavus and enzyme label from the micropore. Add colorless substrate solution and incubate for 10 minutes. All bound enzyme markers convert the substrate into a blue substance. Add the stop solution and read the absorbance value. Compare the unknown concentration sample with the standard absorbance value to obtain the concentration of Aspergillus flavus in the sample.