MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe–rolling circle amplification and Förster resonance energy transfer (pRCA–FRET) were coupled for specific and quantitative detection of miRNA. pRCA–FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.