Objective Aleutian disease, mink enteritis and canine distemper are the three major diseases affecting health of mink. This study intends to establish a multiplex PCR assay for simultaneously detecting of these three viruses. Methods According to the conservative sequences reported in GenBank, three pairs of specific primers were designed to amplify the DNA templates of Aleutian mink disease parvovirus (ADV), mink enteritis parvovirus (MEV), and RNA templates of canine distemper virus (CDV), and optimized the amplifying conditions. Results The specific objective strips of 601 bp (ADV), 205 bp (MEV) and 451 bp (CDV) were amplified simultaneously. The sensitivity test showed that the lowest nucleic acid detection limits were 2.67×10⁴ copies per μL for ADV, 3.02×10⁴ copies per μL for MEV, and 1.72×10⁵ copies per μL for CDV. The results of test of the clinical samples showed that the multiple PCR and single PCR assay were consistent. Conclusions The established multiplex PCR assay in this study can be used to rapidly detect the clinical samples of ADV, MEV and CDV single or mixed infections.