The influence of redox mediators (tetrathiafulvalene (TTF), tetrathiafulvalene-tetracyanoquinodimethane complex (TTF-TCNQ), phenazine methosulfate (PMS), 5,6-diamino-1,10-phenonthroline (5,6-DAP), potassium ferrocyanide (K4[Fe(CN)6]), methylene blue (MB) and toluidine blue (TB)) on the response of amperometric glucose biosensor was investigated in this research. Graphite rod electrode (GRE) modified by adsorbed mediator and glucose oxidase (GOx) cross-linked with glutaraldehyde was served as working electrode. The highest amperometric signals were observed by using TTF and TTF-TCNQ as mediators. The impact of amount of immobilized GOx, an optimal pH region for operation, stability and reproducibility of the analytic signal of designed biosensor using these mediators were investigated and discussed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.