The invention discloses a preparation method and a special culture medium of veterinary D-type clostridium perfringen toxin. The culture medium per 100ml comprises the following components: 1-1.5g soy peptone, 1-1.5g casein peptone, 0.5-0.75g yeast extract, 0.5-0.75g Na2HPO4.12H2O, 1-1.5g dextrin and the balance of water, wherein a pH (potential of hydrogen) value of the culture medium is 8.0-8.5. The preparation method of the D-type clostridium perfringen toxin comprises the steps of inoculating a D-type clostridium perfringen production strain into the culture medium, collecting a culture material, performing centrifugation, and then filtering a supernate. With the adoption of the method, the maximum toxicity can be improved to be 45 times a seedling standard in Regulations on National Veterinary Biological Products, and an output-input ratio can be increased to be 30-225 times that of the original traditional technology. In addition, corresponding serum neutralization titer of a toxoid vaccine prepared by the D-type clostridium perfringen toxin on a rabbit and a sheep is also improved to be 8.3 and 13.3 times a regulation standard respectively.