The invention discloses a preparation method of a neutrophil antigen negative plate. The preparation method comprises the following steps: evenly mixing human heparin anti-coagulation blood with a sodium chloride solution, standing at room temperature, and drawing superstratum plasma; adding the superstratum plasma into a human lymphocyte separation medium containing DextranT500, sufficiently mixing evenly, centrifuging, and drawing a leukocyte solution precipitated at the bottom, so as to obtain a to-be-centrifuged solution; mixing the to-be-centrifuged solution with a LymphoprepTM separation medium, then adding into a centrifuging tube, and placing the centrifuging tube into a horizontal centrifuge machine for centrifuging; drawing a neutrophil solution, so as to prepare cell suspension; and dropwise adding the cell suspension on a clean glass slide, naturally airing, and finally fixing with absolute ethyl alcohol, so as to obtain the neutrophil antigen negative plate. The preparation method solves the problem that the existing method for preparing the neutrophil antigen negative plate has uneven distribution of neutrophils, and the reaction results are easily observed.