The fabrication of glucose oxidase (GOx) enzyme electrodes with controlled alternate enzyme and graphene layers is described. GOx was first modified with pyrene functionalities in order to be self-assembled onto a graphene basal plane via non-covalent π-π stacking interaction. Fluorescence spectroscopy analysis revealed that about 5.4 pyrene functional groups were attached to each GOx and the pyrene functionalized GOx retained more than 76% of the biocatalytical activity compared with the native enzyme. Via alternate layer-by-layer self-assembly of graphene and pyrene functionalized GOx, mono- and multi-layered enzyme electrodes with controlled biocatalytical activity can be easily fabricated. The biocatalytical activity of the as-prepared enzyme electrodes increased with increasing graphene and GOx layers and increased insignificantly when the layers reached four. Such multi-layered enzyme electrodes with controlled nanostructure exhibited reliable application in human serum samples analysis with high detection sensitivity, good stability and repeatability. A broad linear detection limit of 0.2 to 40 mM was obtained.