Brucellosis, a prevalent zoonotic disease in India, causes nonspecific symptoms. Humans contract it via infected animals or by consuming unpasteurized dairy products. This study evaluates the effectiveness of ELISA, Rose Bengal Plate Test (RBPT), and SAT in diagnosing brucellosis from clinical samples. A prospective study was conducted involving 218 patients with unknown fever, arthritis, and neurological symptoms. A detailed history was collected using a pre-designed questionnaire. This study aims to evaluate the clinical diagnostic efficacy of widely used serological assays for human brucellosis, focusing on their sensitivity, specificity, and applicability in clinical settings. Out of 218 clinical samples from suspected brucellosis patients, 129 tested positive for Brucella by either of the serological tests. Male:Female ratio was 1.04:1, and 60% of the patients had a clear history of exposure to cows, buffalo, and goats. ELISA IgG showed the highest positivity rates, identifying 64 (49.6%) cases; ELISA IgM detected 43 (33.3%) cases; the Serum Agglutination Test (SAT) identified 30 (23.2%) cases; RBPT detected 33 (25.5%) cases, and only 14 (10.8%) cases tested positive for ELISA IgG + ELISA IgM. The sensitivities of the ELISA IgG, ELISA IgM, RBPT, SAT, and ELISA IgG + ELISA IgM detection were 69.57%, 46.74%, 35.87%, 32.61%, and 15.22%, respectively, while the specificities were 100%, 100%, 75.40%, 84.92%, and 100%, respectively. ELISA can be used to diagnose human brucellosis reliably and is more sensitive than the SAT and RBPT.