Cytoglobin (Cygb), a recently discovered member of the vertebrate globin family, exhibits a traditional globin fold with a three-over-three α-helical sandwich. The interaction between copper(Ⅱ) ion (Cu2+) and Cygb has been investigated by UV-Vis, fluorescence, synchronous fluorescence and circular dichroism (CD) spectra. Results showed that the absorption intensity of Cygb at 280 nm increased and the intrinsic fluorescence of Cygb was quenched when Cu2+ was added. This fluorescence quenching of Cygb has been proven that it belongs to static quenching. The synchronous fluorescence spectra indicated that there were small changes about the microenvironment of tryptophan residues and tyrosine residues; furthermore, the binding site of Cu2+ is closer to tryptophan residues than tyrosine residues. No obvious change was observed about the secondary structure of Cygb with the addition of Cu2+ from the CD spectra.