Noble metal nanozymes (NMs) are promising alternatives to the fragile glucose oxidase (GOD), however, in all previously reported NMs, O2 consumes electrons from glucose by competing with electrodes, which remarkably limits the Faraday efficiency. The low NM utilization rate and sluggish mass transfer severely limit the electrocatalytic activity. Herein, we report the first Au nanozyme that can catalyze glucose electro-oxidation (GEO) via a distinctive O2 immune pathway with record-breaking mass activity based on apical anchoring and cofactor customization. Strategically, we design a cofactor of lipoic acid (ALA) that can accept electrons from glucose in preference to O2 for Au nanoparticles, and anchor AuNPs/ALA on top of sheared hydrophilic carbon nanotubes (T-SCNT/AuNPs/ALA). Mechanistically, ALA has highly reversible redox activity, and its reduction state is insensitive to O2, thus, it can mediate direct electron transfer between the electrode and AuNPs. In addition, compared to CNT/AuNPs, T-SCNT/AuNPs/ALA has a larger electrochemical surface area, lower charge transfer resistance, and superior hydrophilicity, which are favorable for improving the reaction rate and efficiency. Notably, this strategy can be used to design bilirubin oxidase mimics (T-SCNT/AuNPs/rutin), whose oxygen reduction activity significantly surpasses that of bilirubin oxidase. Consequently, compared to CNT/AuNPs, T-SCNT/AuNPs/ALA boosts the Faraday efficiency of GEO from 50% to 98%, shows a 755-fold increase in mass activity, and enables glucose biofuel cells to offer a 118-fold increase in power density. To the best of our knowledge, this is the first study to achieve a non-O2-interference GEO nanozyme by synergistically regulating the cofactor and catalytic interface and will guide the engineering of demand-specific electrocatalysts.