The invention relates to the technical field of biological detection, in particular to a multiplex PCR detection kit for multiple pathogenic bacteria of waterfowls and application of the multiplex PCRdetection kit. The kit comprises riemerella anatipestifer, escherichia coli, pasteurella and staphylococcus aureus DNA extracting solutions, riemerella anatipestifer ompA-F/R primers, pasteurella kmt-F/R primers, escherichia coli phoA-F/R primers, staphylococcus aureus invA-F/R primers, a 10 * PCR reaction buffer solution, a Pfu PCR mix, and ddH2O. According to the multiplex PCR detection kit formultiple pathogenic bacteria of the waterfowls and the application of the multiplex PCR detection kit, a multiplex PCR detection method capable of simultaneously detecting four waterfowl-derived mainpathogenic bacteria comprising the riemerella anatipestifer, the escherichia coli, pasteurella and staphylococcus aureus is established, the multiplex PCR detection method has the characteristics ofeconomy, rapidness, high efficiency, strong specificity and good repeatability, and can be used for rapidly detecting the waterfowl-derived main pathogenic bacteria.