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Six different electron mediators were immobilized on the activated carbon (AC) anode and their effects on performance of a direct glucose alkaline fuel cell were explored. 2-hydroxy-1, 4-naphthoquinone (NQ), methyl viologen (MV), neutral red (NR), methylene blue (MB), 1, 5-dichloroanthraquinone (DA) and anthraquinone (AQ) were doped in activated carbon (AC), respectively, and pressed on nickel foam to fabricate the anodes. NQ shows comparable performance with MV, but with much lower cost and environmental impact. With NQ-AC anode, the fuel cell attained a peak power density of 16.10 Wm−2, peak current density of 48.09 Am−2, and open circuit voltage of 0.76 V under the condition of 1 M glucose, 3 M KOH, and ambient temperature. Polarization curve, EIS and Tafel measurements were also conducted to explore the mechanism of performance enhancement. The high performance is likely due to the enhanced charge transfer and more reactive sites provided on the anode.