Here, a platform consisting of a fluorescent DNA-functionalized hydrogel was created for the measurement of miRNA-141. In the presence of the target, the closed cytosine-rich loop of the three-segment branched DNA structure is formed as a useful template to create fluorescent silver nanoclusters (AgNCs). Then, aggregation-induced emission of AgNCs occurs in the hydrogel, and enhanced fluorescence is observed. Instead of acrylate-modified DNA, we used unmodified penta-adenine (A5) DNA strands for incorporation into polyacrylamide hydrogel in this method. By raising the concentration of target microRNA 141 (miR-141) under optimal conditions, the silver nanocluster's fluorescence intensities increased linearly from 5 pM to 100 nM, with a detection limit (LOD) of 2 pM. In addition, a selectivity analysis against the target demonstrates that the proposed analytical technique has good selectivity for miR-141. Since the creation of nanoclusters only occurs in the presence of the target sequence, this technique has shown significant potential for sensitive, selective, and label-free miRNA detection for clinical disease diagnosis and biomedical applications.