Electrochemical glucose detection co-catalyzed by bimetallic ions is carried out by introducing Co(II) and Cu(II) into the alkaline solution. The technique avoids the complicated electrode design and catalyst poisoning. By using nitrogen-doped carbon nanotubes (NCNT) loaded on carbon cloth (CC) as the working electrode, the bimetal co-catalytic system achieves a sensitivity of 6,909 mA M−1 cm−2, a detection limit of 0.4 μM (S/N = 3), as well as good selectivity and stability. By performing systematic electrochemical and structural analysis, the mechanism of Co(II) and Cu(II) co-catalysis for glucose detection in the solution is elucidated. Co(II) supplies the dynamic reaction sites and source of active intermediate (CoOOH), while Cu(II) promotes the formation of CoOOH. The results provide mechanistic insights into bimetal co-catalysis of glucose detection and other electrochemical reactions.