Hybrid film of poly(m-phenylenediamine) (PMPD) and Prussian blue (PB) has been formed on a glassy carbon (GC) electrode by one-step electrochemical deposition process for the construction of a highly sensitive and selective glucose biosensor. Due to the nanosized PB particles (ca. 40 nm) in the PMPD-PB hybrid film, the modified GC electrode exhibited good electrochemical activity towards hydrogen peroxide reduction. The PMPD-PB modified electrode responded to hydrogen peroxide in the linear range from 0.0055 mM to 1.0 mM with a detection limit of 0.6 mu M (S/N = 3). The PMPD-PB modified electrode showed good stability in neutral or weak alkaline solutions, and also strong anti-interference ability to other interfering electroactive species such as ascorbic acid and uric acid. Glucose biosensor was further constructed by immobilizing glucose oxidase on the PMPD-PB hybrid-modified electrode surface using gold nanoparticles and chitosan. The present biosensor responded linearly to glucose in the concentration range from 0.025 mM to 0.65 mM with a detection limit of 1.0 mu M (SIN = 3). The response time was 10 s and the sensitivity was 2.54 mA/M. The present biosensor did not show any interference from large excess of ascorbic acid and uric acid. The constructed biosensor had good stability and reproducibility, thus could be applied to the glucose analysis of serum samples in practice. (C) 2012 Elsevier B.V. All rights reserved.