We hereby propose a novel sensitive, specific, and cost-efficient method to detect Ehrlichia canis and Anaplasma platys DNA from canine whole blood samples by multiplex PCR. Blood samples from hemoparasited dogs attending the Veterinary Hospital at the Universidade Federal Rural da Amazônia-UFRA, Belém, Brazil, were collected in tubes containing EDTA. Amplification of E. canis and A. platys 16S rDNA by nested (n) PCR was successfully achieved by using primers specific to the Anaplasmataceae in the first round of PCR, followed by a second round of PCR using E. canis-specific primers in conjunction with A. platys-specific primers. The amplicons obtained were cloned and sequenced, yielding sequences of 478 and 473 bp (including primers) pertaining to regions of the 16S rDNA of E. canis and A. platys, respectively. The protocol we here propose may help to measure the prevalence of canine monocytic ehrlichiosis (CME) and canine cyclic thrompocytopenia, not only in northern Brazil, where there is no data available, but also elsewhere.