The invention relates to a heat-resisting protective agent for duck virus hepatitis live vaccines and a preparation method and application of the protective agent. The method comprises the following steps of: mixing and preparing a freeze-dried protective agent matrix in a certain ratio, performing sterile treatment on the protective agent, dissolving the protective agent components for autoclaved sterilization into double distilled water, and sterilizing for 15 to 30 minutes at the temperature of between 108 and 121 DEG C and dissolving the protective agent components without autoclaved sterilization resistance into double distilled water in a ratio, removing bacteria by using a 0.22mu m filter membrane, mixing the both to form the heat-resisting protective agent, mixing the heat-resisting protective agent and duck virus hepatitis liquid in a ratio of 1: (1-1.2), and performing freeze-drying by adopting a corresponding freeze-drying curve. Compared with the prior art, a good resource is provided for preparation of the duck virus hepatitis live vaccines, the backward status of a domestic vaccine preservation technology is overcome, the live vaccines are preserved for 24 months at the temperature of between 2 and 8 DEG C, and the titers of the vaccines on each feather are kept to be more than or equal to 106.0EID50.
