The invention relates to a deer epidemic hemorrhage competitive enzyme-linked immunosorbent assay kit and a preparation method and a user thereof, the kit comprises an EHDV antigen-coated ELISA plate,a monoclonal antibody IgG-HRP enzyme conjugate, positive serum, negative serum, 20 times of concentrated washing liquid, 10 times of concentrated dilution buffer, a substrate I, a substrate II, a substrate III and stop solution. The preparation method comprises the following steps: a, preparing a deer epidemic hemorrhage virus ELISA-coated antigen, and detecting the safety of the deer epidemic hemorrhage virus ELISA-coated antigen; b, preparing a monoclonal antibody of deer epidemic hemorrhage virus and carrying out preparation and identification on the antibody-horseradish peroxidase (HRP) enzyme conjugate; c, preparing the positive serum and the negative serum; d, preparing the deer epidemic hemorrhage virus antigen-coated ELISA plate; e, preparing the 20 times of the concentrated washing liquid and the 10 times of the concentrated dilution buffer, and preparing the substrate I, the substrate II, the substrate III and the stop solution; and f, assembling the kit. The kit is used fordiagnosis, quarantine, detection and epidemiological investigation of deer epidemic hemorrhage and is characterized by strong specificity, high sensitivity and the like.