Brazilian spotted fever is a serious and lethal illness for humans and is caused by the Rickettsia rickettsii bacteria. In the state of São Paulo/SP (Brazil), the etiological agent of this disease is transmitted by the Amblyomma sculptum tick. It was already shown that horses infected with this bacteria produce a strong immune response and could be important sentinels for the detection of the disease in a proper region. The present investigation performed a serological survey in horses from five farms of Vale do Paraíba, São Paulo state, Brazil, searching for antibodies against, Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali, and Rickettsia bellii. In each farm, ticks were also collected that were taxonomically identified and examined by real-time PCR for Rickettsia spp DNA. Blood samples were collected from 206 horses, and 334 ticks were picked up from these animals from January to December 2017. Eighty ticks wereA. sculptum and 254 Dermacentor nitens. Of the blood samples, 7.3% seroconverted to Rickettsia spp. Of these, 0.97% had a positive serological response to R. bellii. None of the 80 A. sculptum ticks were positive through real-time PCR for Rickettsia spp. Although there was no detection of ticks infected by Rickettsia spp in five farms of Paraíba Valley, the horses presented serological positive reactions against this agent. Thus, further large studies should be conducted in the area targeting hosts and vectors to generate data for control measures of the transmission of Brazilian spotted fever.