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MicroRNAs (miRNAs) play vital roles in a plethora of biological andcellular processes. The levels of miRNAs can be useful biomarkers for cellular events ordisease diagnosis, thus the method for sensitive and selective detection of miRNAs isimperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novelmethod to quantify miRNA expression levels as low as attomolar sensitivity by targetassistedisothermal exponential amplification coupled with fluorescent DNA-scaffoldedAgNCs and demonstrated its feasibility in the application of detecting miRNA in realsamples. The method reveals superior sensitivity with a detection limit of miRNA of 2 aMsynthetic spike-in target miRNA under pure conditions (approximately 15 copies of amiRNA molecule in a volume of 10 μL) and can detect at least a 10 aM spike-in targetmiRNA in cell lysates. The method also shows the high selectivity for discriminatingdifferences between miRNA family members, thus providing a promising alternative tostandard approaches for quantitative detection of miRNA. This simple and cost-effectivestrategy has a potential of becoming the major tool for simultaneous quantitative analysis ofmultiple miRNAs (biomarkers) in tissues or cells and supplies valuable information for biomedical research and clinical earlydiagnosis.