The invention discloses an apolipoprotein E4 ELISA kit and a preparation method thereof, the invention applies the purified human apolipoprotein E4 to prepare an anti-human ApoE4 antibody, and an ELISA plate is coated after the compatible purification. The kit composition includes the ELISA plate coated by the anti-human ApoE4 monoclonal antibody, an enzyme-labeled antibody, ApoE4 standard frozen powder, sample dilution solution, TMB substrate color developing solution, concentration washing liquid and reaction termination liquid. The ApoE4 in the antibody capture standard solution or the sample solution which is fixed on the microporous surface of the ELISA plate is identified by and combined with the enzyme-labeled antibody, so as to form the antibody-ApoE4-antibody-enzyme compound, the absorbance is measured after the reaction and color development of the enzyme and the substrate and the concentration of the ApoE4 in the sample can be calculated by the standard curve. The kit can detect the consistency of the apolipoprotein E4 in human serum, plasma or cerebrospinal fluid, the invention has the advantages of sensitivity, rapidness, simpleness and accuracy, so the invention provides the effective means for in vitro diagnosis and determination of ApoE4 gene type and the gene dosage.