To overcome the safety risks and low utilization efficiency of HO in traditional Fenton processes, production of HO by enzymatic reactions has attracted increasing attention recently. In this study, magnetite-immobilized glucose oxidase (MIG) was prepared to catalyze the heterogeneous Fenton reaction for the removal of trichloroethene from water. The successful immobilization of glucose oxidase on magnetite was achieved with a loading efficiency of 70.54%. When combined with substrate glucose, MIG could efficiently remove 5-50 mg L trichloroethene from water with a final removal efficiency of 76.2% to 94.1% by 192 h. This system remained effective in the temperature range of 15-45 °C and pH range of 3.6-9.0. The removal was slightly inhibited by different cations and anions (influencing degree Ca > Mg > Cu and HPO > Cl > SO ) and humic acid. Meanwhile, the MIG could be recycled for 4 cycles and was applicable to other chlorinated hydrocarbons. The results of reactive oxidative species generation monitoring and quenching experiments indicated that HO generated by the enzymatic reaction was almost completely decomposed by magnetite to produce ·OH with a final cumulative concentration of 129 μM, which played a predominant role in trichloroethene degradation. Trichloroethene was almost completely dechlorinated into Cl, CO and HO without production of any detectable organic chlorinated intermediates. This work reveals the potential of immobilized enzymes for generation of ROS and remediation of organic chlorinated contaminants.