Graphene oxide (GO), having a large specific surface area and abundant functional groups, provides an ideal substrate for study enzyme immobilization. We demonstrated that the enzyme immobilization on the GO sheets could take place readily without using any cross-linking reagents and additional surface modification. The atomically flat surface enabled us to observe the immobilized enzyme in the native state directly using atomic force microscopy (AFM). Combining the AFM imaging results of the immobilized enzyme molecules and their catalytic activity, we illustrated that the conformation of the immobilized enzyme is mainly determined by interactions of enzyme molecules with the functional groups of GO.