A highly performing ECL glucose biosensor was developed by immobilizing glucose oxidase (GOD) onto a membrane modified glassy carbon electrode, which was prepared by using poly(diallyl-dimethylammonium chloride) (PDDA) doped with chitosan. In order to obtain the optimal performance of the ECL biosensor, the composition of modified membranes and a series of measurement conditions were investigated. Under the optimal conditions, this ECL biosensor was able to detect glucose in the range of 0.5-4.0 x 10(4) nM with a detection limit of 0.1 nM (defined as the concentration that could be detected at the signal-to-noise ratio of 3). The relative standard deviation was 0.99% for 5 x 10(-8) mol/L glucose in repetitive measurements in the primary 12 potential cycles. This ECL biosensor offered the effectively improved stability of the electron transfer mediator and exhibited excellent properties for the ultrasensitive and selective determination of glucose with good reproducibility and stability. The present biosensor has also been used to determine the glucose concentrations in real serum samples. The recovery value for the assay of glucose ranged from 96.2 to 107% in the serum samples. The present biosensor displayed both specificity for glucose and retention of signal response even in a complex environment. Therefore, it provided an approach to the sensitive determination of glucose. (C) 2009 Elsevier Ltd. All rights reserved.