The redox-active mediators play pivotal roles in the reactions of the gas-liquid interface and solid-liquid interface of alkaline mediated fuel cells (MedFCs). In this study, Alizarin (Az) is selected to serve as the mediator of alkaline MedFC because of its remarkable reversibility and low permeability. The rapid charge transfer properties and decent alkali stability of Az in alkaline MedFC demonstrate a 1.06 V theoretical open-circuit voltage and a 169.55 mW cm −2 maximum peak power density at 25 °C. Density functional theory calculations and stability analysis reveal an alkali-proof trait of Az due to its uniform charge distribution. Additionally, the alkaline MedFC with 0.2 mol L −1 Az is steadily performed for over 200 h at a discharge current density of 25 mA cm −2 . The utilization of an efficient mediator into a MedFC fundamentally accomplishes the restructuring in its chemistry process. • Alizarin exhibits high electrochemical reaction activity and low ion permeability. • The uniform charge distribution of alizarin results in its alkaline proof. • Alizarin as mediator in alkaline mediated fuel cell reveals excellent performance.