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Abstract DNA‐templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single‐stranded (dC)₁₂when adjacent G‐rich sequences (dG_N, withN = 3–15) were added. The ‘red’ emission of AgNC@dC₁₂with λ_MAX = 660 nm dramatically changed upon the addition of a G‐rich overhang with N_G = 15. The pattern of the emission–excitation matrix (EEM) suggested the emergence of two new emissive states at λ_MAX = 575 nm and λ_MAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA‐based switch that brings AgNC and the G overhang near one another, turning ‘ON’ the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR‐371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR‐371 molecule.