The invention relates to a preparation method of antiserum, and in particular to a preparation method and application of Koi herpes virus antiserum. The preparation method comprises the following steps: selecting healthy Koi as an experimental animal, collecting purified Koi herpes virus antigens, subcutaneously injecting at multiple points of the back of the Koi, when a serum titer reaches a peak value, sampling a great amount of blood from heart or caudal veins, separating serum, measuring a titer, split charging, freezing, and storing. The obtained Koi herpes virus antiserum is used for detecting a KHV (Koi herpes virus) antigen and can be used as positive serum of the KHV antibody detection method. According to the preparation method of the Koi herpes virus antiserum, a preparation method of Koi KHV resistant antiserum is established, the high-titer antiserum is obtained, and the obtained antiserum can be used for detecting the KHV antigen in methods such as IFAT, ELISA and the like and can be used as the positive serum to detect the KHV antibody. A programmed antiserum preparation method is provided for performing the KHV or KHV antibody immunology diagnosis in China, and a finished product immunology diagnosis reagent can be provided.