A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.