DNA molecules have been demonstrated to be good templates for producing silver nanoparticles (AgNPs), with the advantages of well-controlled sizes, shapes, and properties. Revealing the formation kinetics of DNA-templated AgNPs is crucial for their efficient synthesis. Herein, using magnetic tweezers, we studied the reduction kinetics of the Ag+-DNA structure and the subsequent nucleation kinetics by adding NaBH4, L-ascorbic acid, and sodium citrate solutions. At [Ag+] = 0.01 mM, the addition of NaBH4 solution with the same concentration resulted in the restoration of DNA. In contrast, by increasing the [NaBH4]/[Ag+] ratio (r) to 10 and 100, the DNA extension initially decreased rapidly and then increased, indicating nucleation-dissolution kinetics. With AgNO3 solutions of higher concentrations (0.1 mM and 1 mM), direct particle nucleation and growth kinetics were observed by adding a tenfold (r = 10) or a hundredfold (r = 100) amount of NaBH4, which were evidenced by a significant reduction in DNA extension. The reductant dependence of the kinetics was further investigated. Addition of L-ascorbic acid to the DNA-Ag+ solution yielded an increase-decrease kinetics that was different from that caused by NaBH4, suggesting that nucleation was not initially favored due to the lack of sufficient Ag atoms; while sodium citrate showed a weak nucleation-promoting ability to form AgNPs. We discussed the findings within the framework of classical nucleation theory, in which the supersaturation of the Ag atom is strongly influenced by multiple factors (including the reducing ability of the reductant), resulting in different kinetics.