Brucellosis is a zoonotic disease transmitted to humans from infected animals. As a systemic disease, it can harm any organ or system of the host body. Human brucellosis presents with various clinical symptoms, which makes diagnosis challenging. Serological diagnosis of brucellosis is based on ELISA or agglutination tests, which use colorimetry to detect antibodies generated against lipopolysaccharide (LPS) or extracts from whole-cell bacteria. To construct a protein that can specifically recognize Brucella, we analyzed hydrophilicity, accessibility, flexibility, antigenicity, and β-turns using a protein network server. Then, we chose the most abundant immunodominant epitopes of the outer membrane proteins omp31, BP26, omp2b and omp16. Based on the sequences of these major epitopes, fifteen major immunodominant epitopes were selected to construct a synthetic Brucella recombinant multiepitope outer membrane protein (rOmp) gene. This recombinant gene was expressed in E. coli, and the produced protein was purified by Ni-NTA affinity purification. The purified protein was tested in an indirect ELISA assay, demonstrating a high level of sensitivity and specificity. This technique is creating a unique antigen that, coupled with overexpression and low-cost purification, offers a promising diagnosis of both human and animal brucellosis, with the potential to avoid the disadvantages of whole brucellosis-antigen-based assays.