The invention relates to an enzyme-linked immunosorbent assay detection kit and a preparation method thereof. The enzyme-linked immunosorbent assay kit comprises an avidin-coated microplate, a biotin-labeled HbsAb, a biotin-labeled HCV gene recombinant antigen, a biotin-labeled HIV gene recombination antigen, a horseradish peroxidase-labeled HbsAb, a horseradish peroxidase-labeled mouse anti-humanIgG, a horseradish peroxidase-labeled HIV gene recombinant antigen, a substrate solution A, a substrate solution B, a washing concentrate and a stop buffer. The enzyme-linked immunosorbent assay detection kit of the invention can be used to simultaneously detect an HBsAg, an HCV virus IgG antibody and an HIV virus antibody in a sample. The enzyme-linked immunosorbent assay detection kit for bloodsource screening has the advantages of quickness, simplicity, high accuracy, high sensitivity and wide application range. With the detection kit adopted, results can be judged and read with only 2 hours of whole operation time needed. The enzyme-linked immunosorbent assay detection kit is very suitable for blood source screening.