Aflatoxin M₁ (AFM₁), a hydroxylated metabolite of aflatoxin B₁ (AFB₁), possesses the carcinogenic, mutagenic, and toxic activities. To protect the health of animals and humans, it is clear that the need for an effective residue-monitoring programme to detect the AFM₁ residues. In this study, a modified procedure was used to prepare the hapten AFB₁ and AFM₁ derivatives. Then, the prepared antigen AFM₁-CMO-KLH was used to inoculate female Balb/c mice to prepare a sensitive monoclonal antibody (mAb) against AFM₁. After cell fusion and culture several times, the hybridoma cell line, 3D8, which was of the IgG1 isotype, was selected to obtain a highly sensitive mAb. The obtained 3D8 mAb displayed an IC₅₀ value of 64.75 ng L^-1 for AFM₁ and did not exhibit measurable cross-reactivity with other aflatoxins and antibiotics. Based on this mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established that utilizes simple sample preparation and clean-up methods. The decision limit (CCα, α = 1%), detection capability (CCβ, β = 5%), and LOQ value for the AFM₁ matrix calibration method were 24 ng L^-1, 27.5 ng L^-1, and 35 ng L^-1 in the milk matrices, respectively. The AFM₁ recovery ranged from 85.3% to 107.6%. The CVs were less than 13.8%. A positive correlation (r > 0.99) was observed between the ic-ELISA and HPLC-MS/MS results. This ic-ELISA would be a useful tool for screening the AFM₁ residues in milk.