The diagnostic value of enzyme-linked immunosorbent assay (ELISA) was evaluated when blood specimens of 92 patients suspected of brucellosis underwent the ELISA (IgM and IgG), standard tube agglutination (SAT), and 2-mercaptoethanol (2-ME) tests and blood cultures; 38 sera from non-brucellosis patients and 34 sera from blood donors were also subjected to ELISA, SAT, and 2-ME tests. SAT was able to pinpoint only 23 (25%), whereas ELISA confirmed the etiology in 56 (60.9%; P < 0.001) patients with brucellosis, including 31 culture-confirmed cases. The sensitivity and specificity of ELISA were 100% and 71.31%, respectively. Because they were confirmed by ELISA, the diagnosis could never be excluded with SAT in 33 cases. ELISA has been found to be more sensitive in acute (28% higher sensitivity; P < 0.02) and chronic (55% higher sensitivity; P < 0.01) cases. For accurate diagnosis in suspected brucellosis cases detection, we recommend both ELISA IgM and IgG tests. ELISA IgG and 2-ME tests seem to be promising tools in judging prognosis.