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In order to obtain a newly room temperature sintering conductive ink that has great potential applications in printed electronics, especially for some temperature-sensitive substrate, the silver nanoparticles with dodecylamine as the protective agent (Ag/DDA) were prepared, and the effects of protective agent on the insulative–conductive transition time of nanosilver coating at room temperature as well as the stability of nanosilver conductive ink were investigated. The content of the protective agent of Ag/DDA particles was tested by thermogravimetric analysis, and the interaction mechanism between nanosilver and protective agent was studied using Fourier transform infrared spectrometer. In addition, the conductive ink was also prepared with well-dispersed Ag/DDA nanoparticles, and the ink was coated on polyethylene terephthalate and polyimide substrate by Meyer bar (#5). Then, such nanosilver coatings were treated at room temperature to investigate the insulative–conductive transition time. The X-ray diffraction spectra and morphologies of such nanosilver coatings treated at room temperature under different times were also performed. The results showed that the insulative–conductive transition time of the nanosilver coating could be effectively shortened when the protective agent (dodecylamine) is 7.69%. The resulting sheet resistance of the room temperature sintering nanosilver coating after 72 h was 0.15 Ω/sq, which is sufficiently low to be used in printed electronics.