Polymerase chain reaction (PCR) is one of the important molecular biology techniques that helps amplify specific target DNA strands, which can be used in different downstream applications. However, PCR has many obstacles and challenges regarding its sensitivity and efficiency. To overcome those issues, we used metal oxide nanoparticles such as Zinc oxide (ZnO) nano-flower for efficient amplification of foot-and-mouth disease virus (FMDV) serotype O VP1 segment. Incorporating ZnO nanoflowers into the conventional PCR mixture has greatly enhanced the amplified product. The addition of 2 mM of ZnO was able to amplify a relatively low viral load such as 101 and increase the amplified products of higher viral load concentrations. ZnO nanoflowers were also tested with different clinical samples showing superiority over conventional PCR. Overall, the developed nano-PCR can greatly enhance FMDV diagnosis and identification.