Mycobacterium bovis (M. bovis) is a bacterium that causes bovine tuberculosis (bTB) in cattle, and can infect humans. Accurate diagnosis of M. bovis infection is crucial for disease control in animals and in turn, protects people from getting the infection. This study aimed to evaluate the sensitivity and specificity of the so-called GeneXpert MTB/RIF assay in detecting M. bovis in the samples collected from cattle suspects. A retrospective study was conducted, including 50 dairy cows with a history of bTB. The GeneXpert technique, direct microscopy, and sample cultivation were carried out in parallel on all the samples for comparing the diagnostic accuracy of each. GeneXpert showed higher positivity rates compared to the culture. Out of the examined 50 cattle lymph nodes from different slaughtered animals, 30 were GeneXpert-positive, and culture-positive, while 20 were GeneXpert-negative, and culture-negative. The performance of GeneXpert on animal lymph node samples demonstrated reliable detection capabilities. The study highlights the diagnostic potential of GeneXpert in detecting M. bovis infection, and its usefulness in diagnosing and monitoring infections in animal populations. GeneXpert offers improved sensitivity compared to conventional methods, and can aid in the accurate detection of M. bovis infection in clinical samples.