A polymerase chain reaction (PCR) assay was used to amplify a 248-bp sequence (IS1081) of M.bovis DNA using specific primers. Genomic DNA from M. bovis reference strains and M. bo-vis local strains were analyzed and yielded ex-clusively the 248-bp amplified fragment. The sensitivity of PCR detection was determined using different concentration of genomic M. bovis the rapid diagnosis and assists in the control of reference strains DNA. To evaluate the diagnostic ability of the assay for the detection of M. bo-vis in field samples, a total of 54 milk samples from tuberculin reactors cattle  and in clinical samples a total of 31 human sputum samples with suspected pulmonary tuberculosis were tested by PCR,  direct smear and culture examination. in adition DNA ampmlification in non cultivated and cultivated sediment of milk and sputum samples was done and the positive results demonstrated by the presence of a single DNA fragment of 248-bp .Of 57 bacteriological-ly positive samples, 57 were positive by PCR and out 28 of culture-negative samples, gave a positive result by PCR. The PCR showed a sensi-tivity of (100%) and specificity (96.42%) when compared with bacteriological culture method. The specificity and sensitivity of PCR assay in detection of Mycobacterium bovis in field and clinical samples may provide a valuable tool for the rapid diagnosis and assists in the control this zoonosis.