The invention provides an establishing method and application of an animal model of a mycoplasma bovis infected guinea pig, and belongs to the technical field of animal model establishing. The prepared animal model of the mycoplasma bovis infected guinea pig has the sensitive period to mycoplasma bovis of 42 days long, meanwhile effectively simulates the pneumonia symptoms of a mycoplasma bovis infected calf, thus can be used for evaluating the mycoplasma bovis vaccine protection effect, especially the long-term protection effect and has good practical application value.