A comparative genomic analysis of feline coronavirus (FCoV) and feline panleukopenia virus (FPLV) was performed. Based on the conserved regions of the two viruses, specific probes and real-time PCR (qPCR) primers were designed, and a duplex TaqMan qPCR-based assay was established for detecting FCoV and FPLV. The results showed high analytical specificity, and no cross-response with other feline viruses was observed. This method is highly versatile and can be used to detect all FCoV strains stored in laboratories and recombinant plasmids constructed according to the sequences of blank FCoV strains in laboratories. The analytical sensitivity of this method in detecting FCoV and FPLV was as low as 50 copies/μL, which is approximately 20-fold greater than that of conventional PCR. The coefficients of variation (CVs) for the intra- and interbatch coefficients of variation were less than 2%. After 75 clinical samples were tested, the percentage of FCoV- and FPLV-positive samples was 5.34% greater than that of conventional PCR methods, a finding robustly supported by sequencing identification. As validated by clinical samples, the method was sensitive, specific, general, and reproducible and holds great potential for the rapid identification and diagnosis of FCoV and FPLV infections, as well as for epidemiological investigations. KEY POINTS: • One-step duplex TaqMan real-time PCR detection method can detect FCoV and FPLV in clinical samples simultaneously and steadily. • Almost all the currently known FCoV and FPLV strains can be detected. • This method has high sensitivity, specificity and generality.