A label-free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA-21 (mir-21) as the target to examine the desirable properties of this assay. When the target, as a “trigger”, was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the “signal” amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high-affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 at attomole levels. Proof-of-concept evidence has been provided for the proposed cost-effective strategy; thus, this strategy could expand the application of GO-material-based bioanalysis for nucleic acid studies.