A highly selective and accurate on-off ratiometric electrochemical biosensor to detect glucose was proposed based on gold nanoparticles (AuNPs)-glucose oxidase (GOD) nanocomposites as the catalyst for both the oxidation of glucose and the electrochemical reduction of O-2 and the thionine as the inner reference signal for the first time. The reduction peak of O-2 catalyzed by AuNPs-GOD at -0.45 V decreased in GOD-glucose reaction in which the O-2 was consumed gradually to produce H2O2. While, the reduction peak of the produced H2O2 catalyzed by thionine at -0.24 V increased gradually. By using the ratiometric peak current as detection signal, a novel on-off ratiometric electrochemical biosensor for glucose detection was developed and exhibited an acceptable detection limit of 11.66 mu M and a wide linear range from 35.43 mu M to 15 mu M. The biosensor also exhibited high accuracy, high selectivity, good reproducibility and high sensitivity. The ratiometric electrochemical approach not only developed a new method for highly selective and accurate detection of glucose but also provided a good idea for accurate and selective analysis of other analytes. (C) 2017 Elsevier Ltd. All rights reserved.