Abstract： Objective To establish a universal probe for quantitative detection of different mature microRNAs. Methods Based on the most widely used stem-loop framework which was invented by the inventors of stem-loop method,Primer Premier 5.0 software was used to design a universal fluorescent probe and different primer pairs in the stem-loop framework. The probe and the primer pairs were used for quantitative detection of hsa-miR-333-5p,hsa-miR-361-5p,hsa-miR-333-5p,hsa-miR-361-5p,hsa-miR-MiR-423-3p and hsa-miR-486-5p. Results The findings indicated that the universal fluorescent probe could be used for quantitative detection of different mature microRNAs(hsa-miR-143-3p,hsa-miR-145-5p,hsa-miR-124-3p,hsa-miR-339-5p,hsa-miR-361-5p,hsa-miR-423-3p,and hsa-miR-486-5p). Conclusion The universal probe can be used for quantitative detection of RNAi by stem loop method,which can make the quantitative more accurate with lower experimental cost. Theoretically,the probe can be used for fluorescent quantitative PCR detection of all mature microRNAs.