MicroRNAs (miRNAs) have been applied as biomarkers and better detection of their expression profiles plays important roles in early diagnosis of cancers. In this work, a simple dual-signal amplification strategy has been used to construct a novel nanosensor on single Au nanowire electrodes (SAuNWEs) for miRNA-16 detection based on the “signal-on” and “signal-off” features during hybridization/de-hybridization process. The ferrocene-labeled aptamer capture probe (Fc-CP-16) is designed to hybridize with thiolated methylene blue-labeled DNA probe (MB-CP) on SAuNWE to form duplex DNA, and the addition of miRNA-16 can lead to the dissociation of duplex structure due to the highly matched sequences between miRNA-16 and Fc-CP-16. The remaining MB-CP can thus tend to recover its hairpin structure at the presence of Mg 2+ through the hybridization of its complementary sequences. During this hybridization/de-hybridization process, the changes of Fc and MB oxidation peaks can be recorded, and there has a linear relationship between the sum of dual-signal changes (Δ I= Δ I M B+| Δ I F c|) and the logarithm of miRNA-16 concentrations, which can be used to detect miRNA-16. Including miRNA extraction, the dual-signal amplification strategy for miRNA sensing assay was carried out about 2 h for the detection in real samples. This novel nanosensor has small dimension, good selectivity, rapid response and regeneration ability, which can satisfy the need for early cancer marker detection in cells/organelles.