It was reported that graphene oxide (GO), a novel carbon material with unique structural and chemical/physical properties, could improve the performance of polymerase chain reaction (PCR), similar to many nanomaterials. However, GO, with a large lateral size, exhibits poor biocompatibility, and the mechanism of its effect on PCR remains unclear. Using graphene quantum dots (GQDs) that inherit the basic properties of GO, but with nanometer lateral sizes and much better biocompatibility, we systematically investigated their functional roles in PCR enhancement. The overall performance of PCR in terms of its yield, sensitivity, and specificity can be improved with much less GQDs than of GO. It is found that the stacking of the primers on GQDs improves the sensitivity and specificity of PCR through improving efficiency of base-pairing between the primer and the template. The yield of PCR is improved primarily by GQDs via increasing the activity of polymerase, which is tuned by GQDs through chelating magnesium ions with their peripheral carboxylic groups. The adsorption of the polymerase on GQDs affects marginally the activity of polymerase that is different from the proposed function of GO to PCR. The superb performance of GQDs in PCR exhibits their practical potential in PCR, and thus they can be used in biology and medical applications.