Platinum-based bimetallic catalysts exhibit surface atomic rearrangement in various adsorbate environments, which significantly impacts catalysis. A molecular-level understanding of intermediate structures created during catalysis is essential for developing high-performance bimetal catalysts. We show that intermediate Pt–NiO1–x interfacial structures drive the catalytic synergistic effect observed on Pt3Ni nanocrystals. Real-time microscopic observations at ambient pressure show the formation of oxygen-driven Ni oxide clusters on the surface and provide direct evidence of Pt–NiO1–x interfacial structure formation. Spectroscopic analysis, including ambient-pressure X-ray photoelectron spectroscopy and diffuse reflectance infrared Fourier-transform spectroscopy, and catalytic measurements elucidate the role of Pt–NiO1–x interfacial structures and the catalytic reaction mechanism in CO oxidation. Our results indicate that metal-oxide interfacial intermediate structures in bimetal catalysts relate to the catalytic enhancement of the strong metal–support interaction (SMSI) effect.