This study describes the synthesis of Ag nanoparticles using DNA templates with polymorphic structures including the G-quadruplex, the I-motif, and the Duplex and their application for the catalytic reduction of 4-nitrophenol by NaBH4. Interactions between Ag+ and polymorphic DNA were studied through circular dichroism, polyacrylamide gels, and UV spectroscopy. Ag nanoparticles with narrow size distributions were prepared through the reduction of Ag+ by NaBH4 under different DNA templates and Ag+/base ratios. These DNA-templated Ag nanoparticles demonstrated excellent catalytic performance in the reduction reaction of 4-nitrophenol. The rate constants depended on the structure of DNA template, with the decreasing order: I-motif-Ag > G-quadruplex-Ag > Duplex-Ag. The results obtained here suggest a promising pathway to adjust physical-chemical properties of metal nanoparticles through the template of polymorphic DNA.