• A free-standing ERG film with hydrophobic surface realized the direct electrochemistry of laccase. • A faster electron transfer kinetic and a higher bioelectrocatalytic activity towards O 2 reduction with over 10 times’ enhancement in reductive current were achieved at laccase/ERG electrode. • An interfacial hydrophobicity effect was demonstrated for the site-directed capture of laccase immobilization. Direct electrochemistry of oxidoreductase on electrode plays critical roles in the development of enzymatic biosensing and biofuel cells. Herein, a free-standing edge-rich graphene (ERG) film in-situ fabricated on a porous and conductive Si 3 N 4 nanowires template with hydrophobic surface was directly used as a self-supporting electrode for site-directed capture of laccase from Agaricus bisporus . The ERG film possessed abundant edge-rich active sites, high conductivity, and especially hydrophobic surface, which realized the direct electron transfer of the immobilized laccase and its bioelectrocatalysis towards the O 2 reduction. With the comprehensive comparison to hydrophilic ERG, we found that the interfacial hydrophobicity played an important role for the orientated immobilization of laccase. Thereafter a faster electron transfer kinetic (1.32 vs. 0.74 s −1 ) and a higher bioelectrocatalytic activity towards O 2 with over 10 times’ enhancement in reductive current were achieved. This is probably because the hydrophobic region of laccase tends to specifically interact with the hydrophobic surface, allowing the site-directed capture of laccase on the hydrophobic ERG electrode. With an emphasis of the interfacial hydrophobicity effect, these results would not only contribute to an in-depth understanding of the nano-bio interface electron transfer, but also provide a new insight to design high-efficient bioelectrodes for biosensors and biofuel cells applications.