Leptospirosis is considered a bacterial zoonosis of public health importance. It is common in tropical areas, especially in developing countries with scarce health and sanitation resources. This study evaluated the presence of Leptospira spp. in slaughtered bovine in a slaughterhouse in the Midwest region of São Paulo, Brazil, as well as identified positive animals both in serology and by molecular analysis. Biological samples of blood, liver and kidneys from 150 cattle were investigated by the technique of Microscopic Agglutination Test (MAT) and conventional Polymerase Chain Reaction (cPCR). The serological results showed that of the 150 animals, 71 (47.3%) were reactive. The molecular results showed the presence of Leptospira spp. in kidneys of 21 (14%) animals, in liver of five (3.3%) animals, in liver and kidneys in two animals (1.3%) and in blood, in one (0.7%) animal. These results indicate a warning about the health of beef cattle due to the possibility of these animals being the source of infection and the importance of the occupational characteristic of this disease. It was also verified the importance of complementing serological and molecular techniques.