Specific interactions between heavy metal ions and biomaterials have advantages in the sensitive and selective detection of the metal ions. In this study, Cu-demetallated form of Cu/Zn-superoxide dismutase (SOD1) protein was applied as a sensing probe in order to detect Cu(II) ions selectively via surface plasmon resonance (SPR) analysis. Because demetallated metalloproteins have vacant specific metal-binding sites in their own structures, Cu-demetallated SOD1 protein can successfully form stable complex with Cu(II) ion. By applying the properties as a sensing principle, we designed the Cu(II)-selective sensor chip with Cu-demetallated SOD1 protein. When Cu(II) ions were exposed to the fabricated sensor chips, their interaction was quantitatively measured via SPR analysis, and it was proportional to the concentrations of Cu(II) ions. This result reveals successful development of biosensor for the label-free selective detection of Cu(II) ions.