Objective Virus culture, histology, immunology, and PCR have certain shortcomings in the detection of minute virus of mice (MVM), which cannot meet the current needs. Exploring a new method for Rapid and reliable detection of MVM is a major problem that needs to be solved. Methods On the basis of the MVM sequence published by GenBank, we designed primers and probes for Real-time recombinase-aid nucleic acid amplification (RAA) of the NS1 gene, a highly conserved sequence region of MVM, and established a real-time RAA detection method of MVM. We analyzed its specificity, sensitivity, storage stability, and clinical sample validation in comparison with qPCR. Results The sensitivity was 10 copies/ μL, and there was no cross-reactivity with Reo-3, MHV-1, MHV-3, MHV-A59, or MHVJHM viruses. There was no significant deterioration in amplification performance of the RAA stored at room temperature for 20 days. Additionally, the Real-time RAA method was compared with qPCR in 15 positive simulative samples, and the compliance rate was 15/15. Statistical analysis of the correlation between the two method revealed a correlation coefficient of R² =0. 85, which indicates that the two method were significantly correlated. Conclusions The MVM real-time RAA method in this study quickly and effectively detects MVM virus, which also has the advantages of simple operation, strong specificity, and high sensitivity.