The invention relates to a mycoplasma hyopneumoniae culture medium and a preparation method thereof. The mycoplasma hyopneumoniae culture medium consists of a basic culture medium and an auxiliary culture medium, wherein the basic culture medium has major components of Hank's liquid, lactoprotein hydrolysate, a yeast extract and an ox heart extract, sterilization can be performed through autoclaving, and a pollution risk caused by filtration sterilization is greatly reduced; and the auxiliary culture medium comprises pig serum, argenine, cysteine, phenol red solution, penicillium and the like, the pig serum is sterilized through cobalt radiation, and the rest components are mixed with inactivated pig serum after filtration sterilization. The titer of a lapinized attenuated strain of the mycoplasma hyopneumoniae cultured with the culture medium provided by the invention is 109-1010 CCU, the minimum culture time may be 40 h, the generation of old bacteria and aged bacteria is greatly reduced, the usage amount of the pig serum can be as low as 8%, the allergic stress reaction caused by the pig serum is reduced, and the production cost of an enterprise is lowered.