The electrochemical conditioning of amino-carbon nanotubes (CNTs) on a graphene support in an alkaline solution is used to produce [BOND]NHOH as hydrophilic functional groups for the efficient immobilization of bilirubin oxidase enzyme. The application of the immobilized enzyme for the direct electrocatalytic reduction of O2 is investigated. The onset potential of 0.81 V versus NHE and peak current density of 2.3 mA cm−2 for rotating modified electrode at 1250 rpm, indicate improved biocatalytic activity of the proposed system for O2 reduction.