The invention belongs to the field of tuberculosis vaccines, and particularly relates to an antituberculosis vaccine as well as a preparation method and application thereof. Aiming at the problems that an existing vaccine has a poor effect on adults or is unsuitable for hypoimmunity patients and the like, the invention provides the preparation method of the antituberculosis vaccine. The preparation method comprises the following steps: firstly, obtaining a mycobacterium monocell thallus; secondly, interruptedly and circularly irradiating the monocell thallus by adopting rays at low dose to obtain a mycobacterium vaccine, wherein the rays are X-rays, gamma rays or rays generated by an isotope radioactive source Co60; the radiation dose rate lies in that radiation is performed at the rate of10 to 20Gy / min for 8 to 10 times at an interval of 5 to 10 minutes each radiation of 20 minutes. According to the antituberculosis vaccine disclosed by the invention, all antigen characteristics of the thallus are completely retained, stronger specific immune response can be excited more quickly, and effective durable immunity is obtained. The prepared vaccine has the advantages of low toxicity,quick onset and higher safety, and can be used for preventing and treating tituberculosis of immunocompromised patients.