The homology of the sequences, reported and registered in GenBank, of different strains of Avian influenza virus (AIV), Newcastle disease virus (NDV), Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV), was analysed and compared with each other. According to the properties of these viruses, the conservative domain of the M gene for AIV, the F gene for NDV, the 5' non-coding domain end for CSFV and the 2B gene for FMDV were selected for polymerase chain reaction (PCR) amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C (50–60%),18–25 bp in length and Tm (72–85), were analysed using the VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and two or four of the four pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments of different lengths would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Out of 46 pathological samples detected with the multiple PCR, there were 5 AIVs, 7 NDVs, 15 CSFVs and 6 FMDVs. The amplification above was identified with a single PCR. On the other hand, the results corresponded to those of electronic microscopy, haemagglutination (HA) and enzyme-linked immunosorbent assay (ELISA). The method described here is practicable, sensitive, specific, simple and cheap. It could be used for diagnosing AIV, NDV, CSFV and FMDV in different animals.