A considerable effort is currently focused on identifying microRNA (miRNA) biomarkers because they could serve for early disease diagnosis as well as for assessing the prognosis and monitoring the response to treatment. The efficient use of the biomarkers requires precise analysis of miRNAs. This work reports a rapid, sensitive, and selective miRNA assay by coupling the fluorescence quenching of graphene oxide (GO) with site-specific cleavage of an endonuclease. The method is developed by designing a single-stranded probe that carries both a binding region responsible for facilitating the interaction with GO, which induces fluorescence quenching of the 5'-terminus-labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region for specifically recognizing the target and hybridizing with it to form a duplex. The duplex is released from the GO surface through cleavage of RsaI endonuclease, resulting in fluorescence recovery, which shows a trend in the target concentration. The assay can detect down to ∼3.0 fM miR-126 with a linear range of 4 orders of magnitude and has an ability to discriminate the target sequence from even single-base mismatched sequence or other miRNA sequences. Moreover, it can also be used for estimating the miR-126 expressions in cells. The advantage of this assay is that it operates via the detection of the recovered fluorescence signal, which is a combined result of the specific hybridization and the site-specific cleavage, and thus should be impervious to false signals arising due to the nonspecific adsorption of interferants. It could be a great potential tool for selective analysis of miRNAs in cells.