Bovine leukemia virus (BLV) is a retrovirus responsible for lymphoproliferative disorders in cattle. Although infections of BLV in animals are well known, little is known about its capacity to infect humans. This study investigated the presence of anti-BLV antibodies and BLV proviruses in human and cattle samples. An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect anti-BLV antibodies while nested PCR was employed to identify BLV provirus sequences. The overall prevalence of anti-BLV antibodies in human and cattle samples were 12.50% and 16.73%, respectively. When using ELISA as a reference test, sensitivity and specificity for nested PCR were 0.625 and 0.970, respectively. The predictive value of a positive test was 0.862 and the predictive value of a negative test was 0.897. The percentage of cattle correctly classified by nested PCR assay was 89.1%. Nested PCR and Southern blot analysis, using primers specific for BLV gag sequences, revealed that BLV proviruses were detectable in cattle and human samples. Our results highlight the risk of human exposure to BLV and the need for further investigations to determine whether BLV infection poses a health hazard for humans.