The invention relates to a horseradish peroxidase black developer, and a preparation method and an application thereof, and belongs to the technical field of biology. The raw materials of the horseradish peroxidase black developer comprise: 4-CN, DAB, ammonium nickel sulfate, cobaltous chloride, imidazole, polyoxyethylene octyl phenyl ether, boric acid, MgCl2, MnCl2, sodium nitroferricyanide, betaine, gelatin, tetrahydrofuran, acetone, dextran sulfate, 1*TBS and H2O2. The horseradish peroxidase black developer is obtained by uniformly mixing a certain amount of the raw materials. By using the horseradish peroxidase black developer, the color development of a horseradish peroxidase has the advantages of high sensitivity of DAB and good color-difference visual sense effect of 4-CN at the same time, and the color substance is a black deposition with a strong background contrast, and therefore the result determine is convenient.