The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.