A simple microRNA detection method by combining Graphene Oxide (GO) fluorescence quenching with exonuclease III (Exo-III) aided cycling amplification was developed. Three DNA probes, a FAM-containing ssDNA (P-DNA), hairpin probe 1 (H₁) and hairpin probe 2 (H₂), were masterly designed. In the presence of the target, H₁ and H₂ are digested by Exo III because of the target DNA-induced two-step hybridization and a series of single stranded DNAs (ssDNAs) are released; ssDNA and probe P are completely complementary to form double-stranded DNA. This duplex is not easily adsorbed by GO, thus weakening the fluorescence quenching of FAM. In the absence of target miRNA, the ssDNA probes are fully adsorbed on the GO resulting in fluorescence quenching by π–π stacking. This strategy provides a highly sensitive fluorescence detection of microRNA with a limit of detection down to 21.40 pM, and also exhibits good selectivity. The results show that this simple and economical strategy has underlying application value in biomedical research.