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Miniature, self-contained biodevices powered by biofuel cells may enable a new generation of implantable, wireless, minimally invasive neural interfaces for neurophysiological in vivo studies and for clinical applications. Here we report on the fabrication of a direct electron transfer based glucose/oxygen enzymatic fuel cell (EFC) from genuinely three-dimensional (3D) nanostructured microscale gold electrodes, modified with suitable biocatalysts. We show that the process underlying the simple fabrication method of 3D nanostructured electrodes is based on an electrochemically driven transformation of physically deposited gold nanoparticles. We experimentally demonstrate that mediator-, cofactor- and membrane-less EFCs do operate in cerebrospinal fluid and in the brain of a rat, producing amounts of electrical power sufficient to drive a self-contained biodevice, viz. 7 μW cm⁻²in vitro and 2 μW cm⁻²in vivo at …