Here, we describe the standardization of a very sensitive and specific single Plasmodium vivax polymerase chain reaction (PCR) and its usefulness for diagnosis and screening procedures when a Plasmodium falciparum PCR was also utilized. The P. vivax PCR sensitivity threshold was 0.019 parasites per microliter, and a PCR fragment was only detected when P. vivax DNA was present. Among the 11 febrile patients with negative parasitological examination that attended the malaria service of Fundação de Medicina Tropical do Amazonas, we diagnosed one P. vivax malaria by PCR. Among the 286 individuals considered suitable for blood donation, we also detected by PCR an individual with P. vivax malaria, and conversely, we did not detect any malaria infection in blood donor candidates considered unsuitable due to its past malaria history. We conclude that PCR is the method of choice for low-parasitized individuals and could therefore represent a complementary tool to safely rescue blood donor candidates considered unsuitable on the basis of malaria history.