Background <p>Brucellosis continues to pose a significant zoonotic challenge, necessitating the development of improved diagnostic solutions. This study innovatively integrates bioinformatics predictions with prokaryotic expression techniques to create novel diagnostic antigens for the detection of Brucella melitensis.</p> Methods <p>Our study systematically analyzed four outer membrane proteins (OMP10, OMP25, VirB8, and VirB10). B-cell epitope prediction was conducted using the IEDB and ABCpred algorithms. A chimeric antigen was designed by fusing these four proteins in the sequence OMP10-OMP25-VirB8-VirB10. The recombinant protein was expressed in <i>E. coli BL21(DE3)</i> and purified using nickel-affinity chromatography. The diagnostic performance of the purified chimeric antigen was evaluated through indirect ELISA, utilizing 180 brucellosis sera, 20 healthy control sera, and 88 sera from other infections.</p> Results <p>In our study, we successfully predicted and designed a novel recombinant Brucella diagnostic antigen protein, named OMP10-OMP25-VirB8-VirB10, referred to as the ST protein. We assessed its performance as a serological diagnostic biomarker for brucellosis using the indirect enzyme-linked immunosorbent assay. The results demonstrated that the ST protein exhibited a detection sensitivity of 98.33% and a specificity of 85.22%, with an AUC value of 0.9802.</p> Conclusion <p>The ST protein, a fusion of four proteins derived from <i>Brucella</i>, demonstrates significant diagnostic potential in ELISA, offering a novel approach for the accurate diagnosis of brucellosis. This discovery provides an innovative perspective for enhancing early brucellosis screening in endemic regions.</p> Clinical trial <p>Not applicable.</p>