Brucella is one of the world's major zoonotic pathogens and is responsible for enormous economic losses, as well as considerable human disease in endemic areas. The heterogeneous and non-specific clinical symptoms mean that a diagnosis of brucellosis always requires laboratory confirmation. The routine diagnosis of brucellosis involves the conventional serological tests, viz., Rose Bengal plate test (RBPT) and standard tube agglutination test (STAT), but each is associated with certain drawbacks. Therefore, it is always recommended to use a battery of tests in the diagnosis of brucellosis including enzyme-linked immunosorbent assay (ELISA) which is reported to be a sensitive and specific test This study was carried out with 1364 serum samples obtained from persons who were associated with cattle and sheep, based on their occupation from the regions of Telengana, Coastal Andhra and Rayalasema. All these samples were screened and validated using in-house ELISA, in comparison with RBPT, LFA and STAT. A total of 1364 subjects were involved in this study. All of these cases were tested for brucellosis by RBPT, LFA, STAT and ELISA tests. A total of 10.63% of the subjects among all the regions showed positivity with RBPT, the gold standard test, however, with our in-house ELISA, the positivity was 11.22% with sensitivity and specificity of 96.55% and 98.93%. The prevalence brucellosis is more with occupational personnel like veterinarians, paraveterinary staff, dairy farm workers, shepherds who are on close contact with animals, needs to be taken proper protective measures. Brucellosis is categorized as production affecting disease, early diagnosis and treatment will limit the extent of economic loss. ELISA is easy to use and can be performed efficiently with very high sensitivity and specificity compared to many other commercial kits available. © Global Science Publications.