Mycobacterium (M.) bovis, a member of the M. tuberculosis complex (MTC), is a re-emerging, zoonotic agent of bovine tuberculosis whose prevalence probably depends on variations in direct exposure to cattle and ingestion of raw milk. Accurate species differentiation of M. bovis and M. tuberculosis is needed to distinguish between human and zoonotic tuberculosis. This study successfully developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection and differentiation of M. bovis and M. tuberculosis, however showed negative reactions in eight non-tuberculous mycobacteria (NTM) samples and ten other bacterial species. Sensitivity of this assay for detection of genomic M. bovis DNA was 10 $fg/{mu}l$. And this assay successfully detected M. bovis in bovine clinical specimens. In conclusion, the LAMP assay is a simple and powerful tool for rapid detection of M. bovis in both pure bacterial culture and in clinical samples.