Guanidinium chloride-induced unfolding of hemocyanin from Chinese mitten crab ( Eriocheir japonica sinensis), has been studied by UV/vis, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The unfolding of hemocyanin induced by Guanidinium chloride (GuHCl) has been studied by representation of fluorescence data in terms of “phase diagram”. The conformational transition from the native state to a partially folded intermediate of this protein occurs in the range of GuHCl concentration (0–3.5 M), and the transition from the intermediate to the unfolded state of this protein occurs in the range of GuHCl concentration (3.5–6.0 M), indicating that hemocyanin unfolding follows a three-state model under such conditions.