Abstract: Provided are a recombinase polymerase amplification (RPA)-PAND based enterovirus virus typing detection kit and a rapid sensitive detection method, where the detection kit includes a PfAgo protein three gDNAs (g1/g2/g3), MB and its corresponding gDNA (gMB) combination for specifically detection of enteroviruses EV71, CVA6, CVA10, CVA16 and the universal enterovirus (EVU). RPA-HFMD-PAND rapid nucleic acid detection methods for typing detection of common enterovirus type A pathogens EV71, CVA6, CVA10, CVA16 and enterovirus universal EVU are successfully established based on PfAgo protein mediated target detection in combination with an RPA technology. The method has the advantages of high sensitivity, high specificity, low cost, relatively low position targeting restriction, simple reaction system and no aerosol pollution, and can conduct multi-channel nucleic acid detection.