Live cattle export is one of the newest commercial sectors available to Brazilian farmers, but to be exported animals must be free of infectious diseases, especially those prohibited by the countries importing them. In this study, 1,800 bovine sera were collected from animals destined for export in the Brazilian states of Goiás (GO), Minas Gerais (MG), and São Paulo (SP). The sera were tested for bovine brucellosis (BB), paratuberculosis (PTB), enzootic bovine leukosis (EBL), and bovine viral diarrhea (BVD). This was the first study in Brazil to conduct a serological survey for bovine PTB in the states of SP, GO, and MG, and the first survey for BVD in GO. The total percentage of animals positive for each disease was 0.33% (2/1,800) for BB, 16.17% (97/1,800) for PTB, 31.50% (189/1,800) for EBL, and 9.67% (58/1,800) for BVD.