A real-time recombinase polymerase amplification (RPA) assay for pathogenic Yersinia enterocolitica detection was established in this work. According to the sequences of Yersinia enterocolitica pathogenic ail (Attachment Invasion Locus) gene, the specific primer and exo probe were designed and the detection could be finished within 20 mins at 37 ℃. This real-time RPA assay showed a good specificity and the detection limit of the target DNA was 0.1 ng/μL. In stability study, the assay was performed at the DNA concentration of 10 ng/μL, 0.1 ng/μL, 0.01 ng/μL and 0.001 ng/μL in octuplicate. The concentration of 0.01 ng/μL and above could be detected. The relative standard deviation (RSD) of threshold time and maximum fluorescence values of the eight parallels at 0.1 ng/μL DNA were the lowest (≤7.06%). In artificially contaminated powdered milk and beef samples with an initial bacterial concentration of 3 CFU/25 g, the pathogen can be detected using this real-time RPA method after incubation for 20 hours. The detection of 20 different real samples showed that the results of real-time RPA assay were consistent with that of GB 4789.8-2016, which is the national standard of China to detect Yersinia enterocolitica. This real-time RPA assay is helpful for the rapid field inspection at room temperature when conducting qualitative detection of pathogenic Yersinia enterocolitica.