Electrocatalytic ammonia oxidation reaction (EAOR) provides an ideal solution for on-board hydrogen supply for fuel cells, while the lack of efficient and durable EAOR catalysts has been a long-standing obstacle for its practical application. Herein, we reported that the defect engineering via in-situ electrochemically introducing oxygen vacancies (Vo) not only turns the inactive CuO into efficient EAOR catalyst but also achieves a high stability of over 400 h at a high current density of ~ 200 mA·cm⁻². Theoretical simulation reveals that the presence of Vo on the CuO surface induces a remarkable upshift of the d-band center of active Cu site closer to the Fermi level, which significantly stabilizes the reaction intermediates (*NH_x) and efficiently oxidizes NH₃ into N₂. This Vo-modulated CuO shows a different catalytic mechanism from that on the conventional Pt-based catalysts, paving a new avenue to develop inexpensive, efficient, and robust catalysts, not limited to EAOR.