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Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is an airborne disease that endangers wheat during its entire growth period. In this study, the Pst134EA_003354 uncharacterized protein (GenBank: XM_047941824.1) of Pst was used as the target sequence, and the primers PS-RPA-F and PS-RPA-R, as well as the probe PS-LF-probe, were designed for recombinase polymerase amplification (RPA) technology. Flow chromatography was combined with the process to establish an RPA detection method for Pst. This method successfully established visual detection within 10 min under a constant temperature of 39 °C, and the detection results were consistent with those of ordinary PCR analysis. However, it only had high specificity for Pst, and the detection limit was 10 fg/μL. In addition, this rapid method successfully detected Pst from wheat leaves during the field incubation period, indicating substantial benefits for applied use. In summary, the RPA detection method established in this study has the favourable characteristics of high efficiency, simple functionality, and rapid and universal practicability, providing a theoretical basis for the early detection and prevention of Pst., (© 2023. The Author(s).)