Objective To establish a rapid duplex polymerase chain reaction (PCR) assay for simultaneousdetection of Rat coronavirus and Sendai virus. Methods Specific primers were designed based on the N gene of Ratcoronavirus and L gene of Sendai virus. A duplex PCR system was established by optimizing the concentration of primersand annealing temperature, and testing the specificity and sensitivity of this system. This was followed by screening DNAsamples of artificial infections and tissues of experimental animals through our PCR system and comparing it with the ELISAmethod. Results Duplex PCR amplification of Rat coronavirus (168 bp) and Sendai virus (262 bp) was done. Theresults of sequencing of the PCR amplification products were compared with homologous sequences using BLAST functions.The homology of the Sendai virus and Rat coronavirus was 100% and 99%, respectively. The lower limit of detection for Ratcoronavirus and Sendai virus was 1. 56×102 copies/ μL. Specific detection of mouse hepatitis virus amplification resulted ina fragment size that similar to the Rat coronavirus product. The established duplex PCR system was used to detect the DNAsamples of artificially infected Sendai virus, and 30 positive DNA samples were detected. At the same time, in order toverify the applicability of the system, 94 experimental animal lung tissue samples were detected and the results werenegative. Conclusions A rapid PCR assay for simultaneous detection of Rat coronavirus and Sendai virus is successfullyestablished. This duplex PCR assay is specific, sensitive, simple and rapid, and can be used for rapid detection of Sendai virus and Rat coronavirus simultaneously in laboratory animals.