The invention relates to the biological field, and especially relates to a substrate, a method and a kit for in-vitro quantitative determination of the activity of vWF-CP enzyme. The substrate comprises a vWF functional area amino acid sequence. The end N and the end C of the vWF functional area amino acid sequence are respectively combined with a HIS label amino acid sequence and a FLAG label amino acid sequence. The method comprises the following steps: the holes of an ELISA plate are coated with an antibody in advance; and then, the substrate, a sample to be tested, an ELISA antibody, and chromogenic agent are added, and the absorbance of liquid in the holes of the ELISA plate is measured using an ELISA reader. The kit comprises an ELISA plate, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution, or comprises an ELISA plate, an antibody, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution. The substrate, the method and the kit for in-vitro quantitative determination of the activity of vWF-CP enzyme have the advantages of high sensitivity, high accuracy, simple operation and low price, and are suitable for broad patient screening.