Exploring bioelectroanalysis and bioelectrocatalysis in non-aqueous systems are essential for bridging the gap between laboratorial and industrial scale. Bioelectrodes based on carbon nanomaterials, such as carbon nanotubes and graphene, have been designed and fabricated with biocompatible surface functionalities. This review presents recent advances in regulation of a biocompatible microenvironment of enzyme electrodes in non-aqueous systems. We summarize the modification strategies to facilitate electron transfer and promote enrichment of hydrophobic analytes. We focus on the mining and modification for robust oxidoreductases from extremophiles to explore the biosensors in extreme conditions. Challenges and future prospects for bioelectrodes in non-aqueous systems are discussed.