We report the oriented immobilization of [NiFeSe] hydrogenases on both covalently and noncovalently modified carbon nanotubes (CNTs) electrodes. A specific interaction of the [NiFeSe] hydrogenase from Desulfomicrobium baculatum with hydrophobic organic molecules was probed by electrochemistry, quartz crystal microbalance with dissipation monitoring (QCM-D), and theoretical calculations. Taking advantage of these hydrophobic interactions, the enzyme was efficiently wired on anthraquinone and adamantane-modified CNTs. Because of rational immobilization onto functionalized CNTs, the O2-tolerant [NiFeSe]-hydrogenase is able to efficiently operate in a H2/air gas-diffusion enzymatic fuel cell.