Recently, the applications of nanomaterial-assisted polymerase chain reaction (nanoPCR) have received considerable attention. Several potential mechanisms have been proposed, but mainly according to the results of PCR assays under specific conditions and lacking direct and general evidence. The mechanism of nanoPCR has not been elucidated yet. Here, taking gold nanoparticles (AuNPs) as an example, we report the three general effects of AuNPs: (1) AuNPs adsorb polymerase and modulate the amount of active polymerase in PCR, which was directly demonstrated by a simple and straightforward colorimetric assay and the dynamic light scattering measurements. (2) AuNPs adsorb primers and decrease the melting temperatures (Tm) of the duplexes formed with perfectly matched and mismatched primers and increase the Tm difference between them. (3) AuNPs adsorb PCR products and facilitate the dissociation of them in the denaturing step. All these effects were confirmed by addition of a rationally selected surface adsorbent, bovine thrombin, to highly efficiently modulate the surface adsorption of PCR components. These findings suggested that AuNPs should have multiple effects on PCR: (1) to regulate PCR in a case-by-case way via modulating the amount of active polymerase in PCR; (2) to improve PCR specificity in the annealing step via increasing the Tm difference between the perfectly matched and mismatched primers; (3) to improve PCR efficiency via speeding up the dissociation of the PCR products in the denaturing step. Taken together, we proposed the mechanism of nanoPCR is that the surface interaction of PCR components (polymerase, primers, and products) with AuNPs regulates nanoPCR. We further demonstrated that the applications of these findings improve the PCR of the amelogenin genes and Hepatitis B virus gene for genetic analysis. These findings could also provide helpful insight for the applications of other nanomaterials in nanoPCR.