subspecies () is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the gene, for the rapid detection of nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 ( < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R = 0.9012, < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the gene, offers rapid and accurate diagnosis of , with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.