Objective The Standardization of the People’s Republic of China stipulated that Sendai virus (SeV) must be excluded from clean and SPF grade mice. To simplify the detection and improve its efficiency, a novel real-time fluorescence recombinase polymerase amplification assay (real-time RPA) was developed for the detection of SeV in this study. Methods The specific primers and probes were designed against a conserved sequence of the gene encoding the SeV fusion protein, allowing the establishment of this detection method. The specificity, sensitivity, stability and reliability of the assay were evaluated. Finally, the superiority of this detection method was compared with the standard PCR method. Results The assay was highly specific, showing no cross-reactions with six other pathogenic microorganisms such as mouse hepatitis virus. The sensitivity, stability and reliability were acceptable, and this assay was completed within 25 minutes, much faster than the routinely used PCR. Conclusions In this study the SeV real-time RPA detection method is successfully established and found to be a simple, efficient, intuitive and specific detection method.