The invention relates to the technical field of molecular biomedicine, in particular to a PCR (Polymerase Chain Reaction) detection kit for cat and / or dog pathogens, a detection method and application. The kit is used for detecting canine distemper virus, canine influenza A virus, canine parainfluenza virus, canine parvovirus, canine coronavirus, canine rotavirus, canine babesia, canine ascaris, canine Ehrlichia, canine brucella, rabies virus, borrelia burgdorferi, reference gene ACTB, feline herpes virus, feline calicivirus and feline parvovirus. The kit comprises primers and probes of feline coronavirus, feline immunodeficiency virus, feline leukemia virus, feline mycoplasma, feline mycoplasma, feline chlamydia, giardia, toxoplasma, bartonella and reference gene GAPDH, collected DNA and RNA are added into the kit, a real-time fluorescence PCR instrument is adopted for PCR reaction, FAM, HEX, ROX and CY5 fluorescence signals are collected in each cycle, analysis of related pathogens is carried out, and the kit can be used for detecting the feline and the canine. Compared with a traditional detection method, the method has the advantages of higher specificity and higher sensitivity.