Pathogenic variants ofBurkholderia gladiolipose a serious threat to human health and food safety, but there is a lack of rapid and sensitive field detection methods forBurkholderia gladioli. In this study, the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA) was used to detectBurkholderia gladioliin food. The optimized RPA-CRISPR/Cas12a assay was able to specifically and stably detectBurkholderia gladioliat a constant 37 °C without the assistance of large equipment. The detection limit of the method was evaluated at two aspects, the genomic DNA (gDNA) level and bacterial quantity, of which there were 10−3ng/μL and 101CFU/mL, respectively. Three kinds of real food samples were tested. The detection limit for rice noodles, fresh white noodles, and glutinous rice flour samples was 101CFU/mL, 102CFU/mL, and 102CFU/mL, respectively, without any enrichment steps. The whole detection process, including sample pretreatment and DNA extraction, did not exceed one hour. Compared with the qPCR method, the established RPA-CRISPR /Cas12a method was simpler and even more sensitive. Using this method, a visual detection ofBurkholderia gladiolithat is suitable for field detection can be achieved quickly and easily.