To address the limitations of typical hairpin-structural molecular beacons, exploring the ability of a quasi-molecular beacon (qMB) to create label-free fluorescence biosensors is intriguing and remains a challenge. Herein, we propose the first example of modular qMB with the feature of a stimulation-responsive conformation switch to develop an aggregated Ag nanocluster (aAgNC) in a bifurcated DNA scaffold for fluorescently sensing a specific initiator (I*). This qMB was well designed to program four functional modules: I*-recognizable element adopting metastable stem-loop bihairpin structure and two DNA splits (exposed C3GT4 and locked C4AC4T) of aAgNC template that is separated by a tunable hairpin spacer for the customized combination of selective recognition and signaling readout. When presenting I* in an assay route, the specific hybridization induces the directional disassembly of the bihairpin unit, on which the qMB is configurationally switched to liberate the locked split. Thus, the bifurcated parent template pair of C3GT4/C4AC4T is proximal, affording in situ nucleation and clustering of emissive aAgNC. By collecting the fluorescence signal, the quantitative detection of I* is achieved. Benefiting from the ingenious programming of qMB, the recognizing and signaling integration actuates the construction of a facile and convenient fluorescent biosensor featuring rapid reaction kinetics, a wide linear range, high sensitivity, and specificity. This would provide a new paradigm to exploit versatile qMB-based biosensing platforms via stimulation-responsive conformation switches for developing various DNA-scaffolded Ag clusters.