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Feline Calicivirus (FCV) is responsible for a highly contagious disease in domestic cats. FCV may cause multiple symptoms and even death to the infected cats. A simple and cost-effective real-time RPA assay was developed for rapid detection of FCV in clinical samples. In this study, specific primers and probe were designed from the genome of FCV that prevalent in south China. The real-time RPA assay was carried out at 39℃ for 20min before signal analysis by the fluorescence detector. The specificity and sensitivity were thoroughly validated and the results showed that no cross-reaction with irrelevant pathogens were found during the amplification, indicating the good specificity of the new developed real-time RPA assay. RNA standards were constructed and diluted to evaluate the limit of detection. The results showed that the detection limit of the real-time RPA assay could achieve 100 copies/μl, suggesting the high sensitivity of the assay. Additionally, the real-time RPA assay showed excellent performance in clinical sample detection, when compared with a TaqMan qPCR assay. The detection rate of FCV was 38.5% (57/148) for real-time RPA assay and it was a little higher than 37.2% (55/148) of the qPCR assay. Taking all together, the real-time RPA assay had potential application of FCV detection in clinical diagnosis. In conclusion, the new developed real-time RPA assay has provided an alternative strategy for rapid and sensitive detection of FCV in laboratories and animal clinics, especially those with limited facilities.