Abstract: In order to establish a TaqMan real-time quantitative RT-PCR detection method for Canine coronavirus（CCoV）, in the experiment, the whole genome sequences of CCoV downloaded from NCBI were analyzed and aligned, and a pair of specific primers and TaqMan probe were designed in its 3′ UTR conserved region. After the optimization of reaction conditions, a TaqMan real-time quantitative RT-PCR detection method for CCoV was established. The established detection method was used to detect CCoV standard, and the standard curve was drawn; the specificity, sensitivity and repeatability of the detection method were determined. The clinical samples were detected by the established detection method, and the coincidence rate was calculated by comparing with the ordinary RT-PCR detection results. The results showed that the standard curve equation was y=3.25x+10.84, and the correlation coefficient was 0.996 4; it had a good linear relationship in the range of 2.61×10~1-2.61×10~8 copies/μL. Except for the positive test result for CCoV, the detection for Canine parvovirus, Canine distemper virus, Canine adenovirus and Canine parainfluenza virus was all negative. The lower limit of detection was 2.61×10~1 copies/μL; the coefficient of variation between batches and within batches was less than 2%. TaqMan real-time fluorescence quantitative RT-PCR detected 9 positive clinical samples, while ordinary RT-PCR detected 5 positive clinical samples. The coincidence rate of the two detection methods was 91.66%. The results suggested that the established CCoV TaqMan real-time quantitative RT-PCR method had high sensitivity, strong specificity and good repeatability, and could be used for clinical detection of Canine coronavirus disease.