The invention discloses a mycoplasma bovis medium and a preparation method thereof. The mycoplasma bovis medium comprises a basic medium and an auxiliary medium through mixing and is prepared from Friis media premix, sodium pyruvate, a fresh yeast extract liquid, glucose, phenol red, deionized water, L-glutamine, L-cysteine, lactoalbumin hydrolysate, transferrin, insulin, penicillin and a small amount of horse serum. The medium not only can reduce the use amount of serum, but also remarkably increases the titer of living bacteria of mycoplasma bovis and lays a foundation for preparation of high quality vaccines of mycoplasma bovis.