The invention discloses a combination of multiple PCR primers and probes for detecting pathogens and its application. The present invention uses influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus (distinguishing type I, type II, type III), enterovirus, dengue virus, eastern equine encephalitis virus, western equine encephalitis virus Encephalitis virus, Venezuelan equine encephalitis virus, forest encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, Nipah virus, and Hendra virus were developed and designed to simultaneously detect the above pathogens Based on the combination of multiple PCR primers and probes, corresponding gene chips and detection kits were developed. The primer and probe combination designed and obtained by the invention has strong specificity, wide detection range and good result accuracy, which not only helps the prevention and control of infectious pathogens, but also can be used for inter-laboratory quality assessment of corresponding pathogens.