This study reports a design of experiment methodology to investigate and improve the performance of glucose oxidizing enzyme electrodes. Enzyme electrodes were constructed by co-immobilization of amine-containing osmium redox complexes, multiwalled carbon nanotubes and glucose oxidase in a carboxymethyldextran matrix at graphite electrode surfaces to provide a 3-dimensional matrix for electrocatalytic oxidation of glucose. Optimization of the amount of the enzyme electrode components to produce the highest current density under pseudo-physiological conditions of 5 mM glucose in saline buffer at 37 degrees C was performed using response surface methodology. A statistical analysis showed that the proposed model had a good fit with the experimental results. From the validated model, the addition of multiwalled carbon nanotubes and carboxymethyldextran components was identified as major contributing factors to the improved performance. Based on the optimized amount of components, enzyme electrodes display current densities of 1.2 +/- 0.1 mA cm(-2) and 5.2 +/- 0.2 mA cm(-2) at 0.2 V vs. Ag/AgCl in buffer containing 5 mM and 100 mM glucose, respectively, largely consistent with the predicted values. This demonstrates that use of a design of experiment approach can be applied effectively and efficiently to improve the performance of enzyme electrodes as anodes for biofuel cell device development. (C) 2015 Elsevier B.V. All rights reserved.