The invention discloses a pullorum staining agglutination antigen as well as a preparation method and an application thereof, and belongs to the technical field of veterinarian diagnosis. Salmonella pullorum SP7220, SP7701, SP8441 and SP9905 are subjected to recovery and passage respectively to form a seed bacterium liquid, then the seed bacterium liquid is inoculated with a solid culture medium for proliferation, after inactivation with formalin, the concentration of the bacterium liquid is adjusted with turbidimetry, finally, the bacterium is fully and evenly mixed and packaged after a crystal violet solution is added for staining, and the pullorum staining agglutination antigen is prepared. The technological method is simple, reasonable, scientific, stable in production and low in cost, the selected production stains of the pullorum staining agglutination antigen are good in antigenicity and low in mutation rate, the prepared pullorum staining agglutination antigen product has the advantages of high sensitivity, high specificity, quickness in diagnosis and clearness in agglutination image, the staining agglutination antigen is relatively ideal for pullorum detection.