The invention belongs to the technical fields of molecular biological techniques and clinical tests, and particularly discloses a nosocomial infection multiple causative agent parallel detection gene chip and a preparation method and application thereof. The gene chip can be used for rapidly and sensitively detecting fourteen medical treatment infection typical causative agents such as acinetobacter baumannii, enterobacter cloacae, enterococcus faecalis and the like by using one reaction, and the detection data of coinfection of yeast and bacterium which cannot be provided by a traditional amplification method is offered. Furthermore, the gene chip also comprises a methicillin resistance target spot, thus realizing further detection of staphy lococcus infection, therefore, a clinician can realize which causative agent a patient is infected in one or two hours, and realize whether the patient has drug resistance, so as to decide whether the patient needs antibiotics or which antibiotics is needed, so that the gene chip has a greater clinical guiding significance.