The invention provides a swine mycoplasma hyopneumoniae culture medium and a preparation method and application thereof and belongs to the technical field of bioengineering. The swine mycoplasma hyopneumoniae culture medium is prepared from a basal culture medium and an auxiliary culture medium, which are mainly prepared from ingredients such as MEM, beef extract powder, yeast leachate powder, lactoalbumin hydrolysate, gastric mucin, an arginine solution, pig blood serum and chicken blood serum. The swine mycoplasma hyopneumoniae culture medium is prepared through subjecting the basal culture medium and the auxiliary culture medium to sterile treatment, then, carrying out volume determination by using injection water, and adjusting the pH value of the solution. The culture medium provided by the invention is low in blood serum content and is applied to the preparation of vaccine antigens, the growth speed of swine mycoplasma hyopneumoniae is high, the culture cycle is short, the fungus content of a semi-finished product fungus solution is high, the production cost is low, and the prepared vaccines are good in immunization effect and low in side reaction occurrence probability, so that the culture medium is suitable for being industrially produced on a large scale.