The invention discloses a preparation method of an agkistrodon acutus venom detection kit based on an improved double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method, which comprises the following steps: step 1, primary purification: primarily purifying horse anti-agkistrodon acutus IgG in horse anti-agkistrodon acutus plasma by adopting a saturated ammonium sulfate method; 2, repeatedly purifying, further performing chromatographic purification on the horse anti-agkistrodon acutus IgG by adopting affinity chromatography, collecting an antibody corresponding to a target peak, and performing column chromatography purification again to obtain relatively pure horse anti-agkistrodon acutus IgG; 3, coating an antibody: taking a part of the antibody purified in the step 2 as a coating antibody, and coating the coating antibody in a 48-pore plate; step 4, labeling an antibody, taking a part of the antibody purified in the step 2 to react with horse radish peroxidase (HRP) to obtain a horse radish peroxidase labeled antibody (HRP-IgG), and taking the HRP-IgG as a sandwich antibody; and 5, packaging the kit, sealing the 48-pore plate coated in the step 3 in vacuum, and putting the 48-pore plate and the sandwich antibody in the step 4 into the kit for later use, so that the kit is prepared and can be used for detecting whether human or animal serum contains agkistrodon acutus toxin or not.