The binding interaction between bovine hemoglobin (BHb) and a novel two-dimensional carbon nanomaterial, graphene oxide (GO), has been investigated in this work. It is found that GO strongly disturbs the secondary structure of BHb by forming BHb-GO aggregates. The binding affinity between the GO and BHb is shown to be mainly from non-covalent interactions including hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions. In addition, two possible binding modes are proposed, insert binding mode and surface binding mode, as shown by molecular modeling. Our findings also show that the existence of GO can significantly prevent the non-enzymatic glycosylation of BHb and decrease the thermal stability of the protein. This work elucidates the effects of the binding interactions of GO with BHb on some biological properties and functions of heme protein. (C) 2016 Elsevier B.V. All rights reserved.