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The vitamin B12 molecule has long fascinated chemists because of its exclusive complex structure and unusual reactivities in biological systems. In order to achieve a better understanding of the structural attribute of the Vitamin B12 molecule when it interacted with metal, in the present paper, the vitamin B12 molecules adsorbed on variation of copper electrode potential from 0 to -1.0 V was studied by surface-enhanced Raman spectroscopy (SERS). An excellent SERS substrate was obtained with insitu electrochemical oxidation-reduction cycle (ORC), and its surface roughness was characterized by atomic force microscope (AFM). Assignments of Raman peaks observed by normal Raman spectrum (NRS) and SERS spectra of vitamin B12 molecule were given based on previous literatures. It was found that the potential-dependent relative intensity changed in SERS spectra which depended on the vitamin B12 molecular orientation with respect to the copper surface according to the surface selection rule (SSR). It was concluded that the corrin ring was adsorbed in tilt form on copper surface and the Co-CN group was farther away from the copper surface at higher potentials. With the decrease in potential, the tilt angle between the corrin ring and copper surface became smaller, then the Co-N group and 5,6 dimethylbenzimidazole group got close to the copper surface. The results offered an important structural attribute of vitamin B12 molecule when it interacted with copper electrode for the first time, and supplied a meaningful reference for the electrochemical bioactivity of the vitamin B12 molecule.