In this study, two ortho-quinoidal compounds, 1,10-phenanthroline-5,6-dione (PD) and 9,10- phenanthrenequinone (PQ), were examined as electron transfer mediators suitable for amperometric glucose biosensors. The dependences of the electrochemical responses of PD- and PQ-based amperometric glucose biosensors on varied concentrations of glucose were investigated under aerobic and anaerobic conditions. The PD-modified graphite rod (GR) electrode revealed a current response seven times higher than that of the PQ-modified GR electrode. The reactivity indices of orthoquinoidals assessed by means of B3LYP functional method applying 6-311G(D) basis set showed that the electronaccepting potency for PD was markedly higher as compared with that of PQ. Compared to PQ, considerably higher reactivity of PD has been defined in the reactions with NADP+- ferredoxin reductase (FNR, EC 1.18.1.2) as a model singleelectron transfer FAD-dependent enzyme, which provided an additional evidence for PD as a more efficient mediator compared to PQ. This study illustrates that PD can be applied as a redox mediator for glucose oxidase and it could be more suitable for a reagent-less biosensor design than PQ. © Springer-Verlag Berlin Heidelberg 2014.