Dark or weak-emissive DNA-harbored silver nanoclusters (AgNCs) can be remarkably lighted up when approaching to guanine bases. The resultant bright AgNCs acting as a fluorescent reporter are fascinating in biosensing. To explore the applicable guanine-enhanced emission of AgNCs for biosensing microRNA-155 (miR-155) as a model, here we designed a unique stem-loop hairpin beacon (HB) encoding with an miR-155-recognizable sequence and a AgNCs-nucleable template, as well as a hairpin helper tethering a partially locked guanine-rich (15-nt) tail (G15H), while two identical cytosine-rich segments were inserted in HB and G15H to merge for folding/unfolding of i-motif at changed pHs. Initially, the intact clusters populated in HB (HB/AgNCs) were almost nonfluorescent in a buffer (pH 7.0). Then, miR-155 was introduced to trigger a repeated hairpin assembly of HB and G15H by competitive strand displacement reactions at decreased pH 5.0 within 10 min, consequently generating numerous duplex DNA constructs (DDCs). With the resultant template of pH-responsive i-motifs incorporated in DDCs, their folding at pH 5.0 brought the proximity of unlocked G15 overhang to the clusters in a crowded environment, remarkably lighting up the red-emitting fluorescence of HB/AgNCs (λem = 628 ± 5 nm) for amplified signal readout. About 3.5-fold enhancement of quantum yield was achievable using different variants of i-motif length and G15 position. Simply by adding OH-, the configuration fluctuation of i-motifs was implemented for switchable fluorescence biosensing to variable miR-155. Based on a one-step amplification and signaling scheme, this subtle strategy was rapid, low-cost, and specific for miR-155 with high sensitivity down to 67 pM.