Nickel-binding proteins play an important role in the biological processes and can also be utilized in several fields of biotechnology. This study was focused on analysing the nickel-binding proteins from the blood sera of humans (Homo sapiens), cattle (Bos taurus), sheep (Ovis aries), red deer (Cervus elaphus), mouflon (Ovis orientalis), fallow deer (Dama dama), horses (Equus ferus caballus), pigs (Sus scrofa domesticus), wildboars (Sus scrofa), brown bears (Ursus arctos) and pheasants (Phasianus colchicus). The presence of higher abundance proteins in the blood serum, such as albumins, may mask the detection of lower abundance proteins. The samples were depleted from these higher abundance proteins to facilitate the detection of those with lower abundance. For the characterization of these proteins, nickel cations bound to tetradentate ligand nitrilotriacetic acid(Ni-NTA)immobilized on agarose beads were incubated with animal sera to capture nickel-binding proteins and subsequently the proteins were eluted and fractionated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed a set of nickel-binding proteins with various molecular weights within different animal species. A unique ~42 kDa nickel-binding protein in the brown bear serum, which was not present in any of the other species, was further characterized and identified by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). This protein was identified as ahaptoglobin-like protein. This result may provide some valuable clue for the physiological difference in the metal binding proteins in the serum of Ursus arctos and other animals.