Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022–2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 102 copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19–1.31% and inter-assay variability of 0.10–0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.
