An ELISA assay was performed to detect antigens of Brucella melitensis directly in the blood of patients affected by Brucellosis. Disposable polystyrene microtiter plates were coated with rabbit immunoglobulins anti-Brucella melitensis antigens and then incubated with sera of Brucellosis patients and sera of not infected normal subjects as a control, to standardize the conditions of the different steps of the assay. The level of the blood-containing Brucella antigen bound to the plate was measured by addition of anti-Brucella melitensis antiserum conjugated with alkaline phosphatase followed by incubation with the specific enzyme substrate. Sera from 9 Brucellosis patients not undergoing therapy were tested by this ELISA assay, and all showed values significantly higher than the control. A pool of 96 sera from normal subjects not infected with Brucella melitensis was used as a negative control. In addition, a different group of 8 sera from patients with Brucellosis undergoing therapy were also analyzed, but no difference in the ELISA value was observed between the two groups with or without therapy. The ELISA assay described in this paper could be a reproducible, sensitive and suitable test to detect Brucella-antigens in the blood of Brucellosis patients and it could be used in addition to the more common methods for a more thorough diagnosis of Brucellosis.