The invention discloses a mycoplasma bovis attenuated strain and application thereof. The mycoplasma bovis attenuated strain is prepared by the following steps: A. a mycoplasma bovis virulent strain is separated and determined: by pathogeny separation culture and PCR (Polymerase Chain Reaction) detection, the pathogeny is determined to be mycoplasma bovis; B. the mycoplasma bovis virulent strain is cultured: the mycoplasma bovis which is obtained by separation is inoculated with a liquid drug culture medium, and is then cultured in an incubator, wherein the culture medium becomes bright yellow from red, and the previous generation of bacterium liquid of 1mul is taken for inoculating PPLO (pleuropneumonia-like organism) culture medium and culturing; C. the full attenuation of mycoplasma bovis can be shown by the experiments, and by the detection of morphology, and the generation 150 of strain Mbov HB0801-150.2 cultured by mycoplasma bovis Mbov HB0801 can be verified by the experiment result; and D. the preservation number of the mycoplasma bovis Mbov HB0801-150.2 is CCTCC M20111102. The invention has good vaccine development prospective, wide clinic application value, low cost and small irritant to animals, and can produce huge economic benefits and social benefits.