Abstract We demonstrate the construction of glucose sensors employing pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQGDH) from Acinetobacter calcoaceticus and glucose oxidase (GOD) from Aspergillus nigar coupled with Escherichia coli soluble cytochrome b 562 (cyt b 562) as electron acceptor. PQQGDH and GOD do not show direct electrochemical recycling of the prosthetic group at the electrode surface leading to a corresponding current signal. We constructed PQQGDH and GOD electrodes co-immobilized with 100-fold molar excess of cyt b 562 and investigated the electrochemical properties without synthetic electron mediators. PQQGDH/cyt b 562 and GOD/cyt b 562 electrodes both responded well to glucose whereas no current increase was observed from the electrode immobilizing enzyme alone. The detection limits for the PQQGDH/cyt b 562 and GOD/cyt b 562 electrodes were 0.1 and 0.8 mM, respectively, and their linearity extended to over 2 and 9 mM, respectively. These results demonstrate that a sensor system can be constructed without a synthetic electron mediator by using a natural electron acceptor. Furthermore, we have demonstrated the potential application of cyt b 562 in direct electron transfer type sensor systems with oxidoreductases whose quaternary structure do not contain any electron transfer subunit.