Multiplex PCR is a new molecular biology detection technology, which is highly efficient, rapid, convenient, specific, and sensitive. It overcomes the shortcomings during amplification in conventional PCR and enables the synchronous detection of multiple pathogens in a single reacting system. It can thus provide new clues for research on differential diagnoses and the genotyping of resistance genes in a clinical context. This paper summarizes the research background and principles of multiplex PCR, and its use for the detection of viruses, bacteria, and parasites as well as microbial resistance in experimental animals. This paper provides a reference for the use of multiple PCR technology for detecting pathogens causing experimental animal epidemics and the establishment of method to combat them.