The aggregation behaviors of silver atoms modulated by polymorphic DNA templates involving i-motif, G-quadruplex, and the Watson-Crick duplex, were investigated by using the density functional theory (DFT) calculations, combining with the experimental characterizations of CD, UV, fluorescence measurements and TEM, in order to understand the reason in the molecular level that polymorphic DNA templates affect the fluorescence emitting species of Ag nanomaterials. First, the affinity sites of silver ions on different DNA templates were analyzed by using DFT calculations, and the conformational variations of DNA templates caused by silver ions and atoms were disclosed. Second, the aggregation behaviors of silver atoms constrained by the polymorphic DNA templates were studied by DFT modeling, and distinct fluorescence property of nanosilvers templated by polymorphic DNA were evaluated using the time-dependent DFT calculations. It is illustrated that with the DNA template adopting i-motif or the duplex the silver atoms tend to aggregate inside the encapsulated spaces of nucleobases, and the formed silver nanoclusters are positively charged with high fluorescent spectral features; whereas with the template of the G-quadruplex the silver atoms are preferential to aggregate outside of the G-tetrad, which results in the formation of larger silver crystals without fluorescence property. The results obtained here are useful to explore the nucleation and growth mechanism of silver nanomaterials regulated by the structure-specific DNA templates, which is important to rational design of desirable fluorescent emitters for sensing in the field from biology to nanoscience.