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<i>Mycobacterium bovis</i> (<i>M. bovis</i>), the microorganism responsible for bovine tuberculosis (bTB), is transferred to people by the ingestion of unpasteurized milk and unprocessed fermented milk products obtained from animals with the infection. The identification of <i>M. bovis</i> in milk samples is of the utmost importance to successfully prevent zoonotic diseases and maintain food safety. This study presents a comprehensive description of a highly efficient molecular test utilizing recombinase-aided amplification (RPA)–clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) 13a–lateral flow detection (LFD) for <i>M. bovis</i> detection. In contrast to ELISA, RPA–CRISPR–Cas13a–LFD exhibited greater accuracy and sensitivity in the detection of <i>M. bovis</i> in milk, presenting a detection limit of 2 × 10⁰ copies/μL within a 2 h time frame. The two tests exhibited a moderate level of agreement, as shown by a kappa value of 0.452 (95%CI: 0.287–0.617, <i>p</i> < 0.001). RPA–CRISPR–Cas13a–LFD holds significant potential as a robust platform for pathogen detection in complex samples, thereby enabling the more dependable regulation of food safety examination, epidemiology research, and medical diagnosis.