Lignocellulose pretreated by pyrolysis can be transformed into clean energy (such as bioethanol) via microbial fermentation, which is significant for waste recycling, environmental protection, and energy security. However, acids, aldehydes, and phenols present in the bio-oil exert inhibitory effects on microorganisms, which challenges the downstream utilization and conversion of lignocellulosic pyrolysate. In this study, we constructed a microbial electrolysis cell system for bio-oil detoxification and efficient ethanol production (C2H5O) using evolved Escherichia coli. In the bio-oil media with electricity, E. coli-H exhibited significant levoglucosan consumption and ethanol production abilities compared to the control. Finally, 0.54 g ethanol/g levoglucosan was produced by E. coli-H in the undetoxified bio-oil media with 1.0%(w/v) levoglucosan, reaching 95% of the theoretical yield. This design aimed to solve bioethanol production and utilization challenges, as evident in related studies. This research provides a practical method for bioethanol production from lignocellulosic substrates and a scientific basis and technical demonstration for its industrialized application.