INTRODUCTION: Brucellosis, an infectious zoonotic disease caused by members of the genus METHODS: In this study, ErfK, YxeI, and EntF were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera from bovines and sheep diagnosed with brucellosis were analyzed using indirect ELISA with these proteins. RESULTS: For bovine serum, the combined protein group (ErfK + YxeI + EntF) and YxeI demonstrated the highest diagnostic accuracy of 94.23% and 93.58%, respectively. Meanwhile, the combined protein group showed the strongest ability to detect CONCLUSION: The findings of this study suggest that Tat substrate proteins serve as promising candidate antigens with significant potential value for the clinical diagnosis of brucellosis.