Dynamic DNA assembly, operated in an autonomous and reconfigurable manner by controlling the kinetics of strand displacement reactions (SDR), is an ideal approach to amplify the fluorescent signals for molecular diagnostic and imaging. Herein, we for the first time have demonstrated an enhancement of fluorescence intensity of DNA/Ag nanocluster-based beacons by the modulation of SDR. This is a new DNA/Ag NCs fluorescence light-up system through the use of the enhancer of G-rich overhang. Such a sensing system can be used to develop a DNA/Ag nanocluster-based beacon for the fluorescent detection of nucleic acid and thrombin with high selectivity and sensitivity, in which the detection sensitivity could be further enhanced through additional Exo III based amplification.