Objective To construct armored RNA reference material containing target RNA of group A Rotavirus (RV) based on Qβ bacteriophage, to determine the value and to test the homogeneity and stability of such material. Methods DNA fragment named QβSNRV which contained maturase-coding gene, capsid protein-coding gene, packing site of Qβ bacteriophage, detection target sequence of group A Rotavirus and multiple clone sites from the 5′ end to the 3′ end was synthesized, and then subcloned into pET-28a (+) expression vector. The recombinant plasmid was pET-QβSNRV that was identified by enzyme digestion and sequencing, and then transformed into Escherichia coli BL21 (DE3) competent cells and expressed. The expressed product, virus-like particles of Qβ bacteriophage containing RNA of RV, named AR-RV, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). AR-RV was centrifuged and purified by CsCl density gradient ultracentrifugation and sephacry gel chromatography. The morphology of AR-RV was observed by transmission electron microscopy. The valuation, homogeneity and stability of AR-RV were tested according to the GB/T 15000.3-2008. Results SDS-PAGE analysis showed that the molecular mass of the expressed protein product was about 14.1 kDa. The virus-like particles of AR-RV, 25 nm in diameter, with typical morphology could be observed under electron microscope. AR-RV samples prepared in this study were valued as (1.02±0.3)×10⁷ copies/μl and behaved well in the homogeneity test, F=0.66＜F0.05(9,0). The stability test indicated that the sample was stable at 37 ℃ for 15 days, 25 ℃ for 15 days, 4 ℃ for 50 days, -20 ℃ for at least 270 days, -80 ℃ for at least 360 days with no significant decrease. Conclusion The group A Rotavirus armored RNA based on Qβ bacteriophage was successfully prepared and had good uniformity, stability and high copy number. This method could supply with a good and biologically safe reference material candidate for the Rotavirus virus RNA detection.