MicroRNA (miRNA) sensing strategies employing rolling circle amplification (RCA) coupled with the hairpin DNA (HD) probe-mediated FRET assay have shown promise, but achieving rapid, sensitive, and specific detection of target miRNA remains a challenge in clinical diagnostics. Herein, we incorporate PstI endonuclease cleavage (PEC) into a conventional RCA-based HD probe FRET assay to develop an effective and feasible method. Long single-stranded RCA products are synthesized from miRNA-21 loaded on a circular dumbbell DNA, and the resultant RCA products self-assemble to generate long HD structures with double-stranded stem regions that are specifically recognized and cleaved by PstI endonucleases when incubated with PstI enzymes. This releases large amounts of short single-stranded DNA fragments that hybridize and open to the complementary loop-stem regions of HD probes labeled with FAM at one end and BHQ-1 at the other, resulting in a reduction in FRET efficiency. This assay achieves a 39.7 aM detection limit for target miRNA-21, approximately 37-fold higher than that of the conventional assay (1.5 fM). Moreover, quantitative detection is possible in a wide range from 1 aM to 1 pM within 90 min with high sequence specificity. We demonstrate the assay with the detection of target miRNA-21 in total RNA extracted from MCF-7 cancer cells.