MicroRNAs (miRNAs) are important biomarkers for disease diagnosis and prognosis. Accurate and robust detection of miRNAs greatly facilitates the early diagnosis and progress assessment of certain cancers. The current miRNA detection based on the sole fluorescence intensity at a certain emission wavelength is prone to instrumental and environmental interference, reducing the reliability of detection. Here, we innovatively developed hairpin DNA-templated silver nanoclusters (DNA-AgNCs) as a ratiometric fluorescent probe for miR-141 detection. The native DNA-AgNC probe could emit relatively strong green fluorescence (λmax at 555 nm) and weak red fluorescence (λmax at 640 nm) upon excitation at 465 nm and 575 nm, respectively. In the presence of the target miR-141, the green emission of the DNA-AgNCs decreased, with the red one increasing concurrently. The ratio of the fluorescence intensity between the red and the green emission was linearly related to the concentration of miR-141, providing a simple approach for a miRNA assay with relatively higher sensitivity and a single nucleotide specificity in miR-141 detection.