A third-generation biosensor based on HRP and a Sonogel-Carbon electrode has been fabricated with the aim of monitoring hydrogen peroxide in aqueous media via a direct electron transfer process. The redox activity of native HRP, typical of thin-layer electrochemistry, was observed. The charge coefficient transfer,alpha, and the heterogeneous electron transfer rate constant, k(5), were calculated to be 0.51 +/- 0.04 and 1.29 +/- 0.04 s(-1), respectively. Topographic study by atomic force microscopy(AFM) shows that the enzyme may have been introduced inside the ionic cluster of the Nafion. The immobilized HRP exhibited excellent electrocatalytical response to the reduction of H2O2 and preserved its native state after the immobilization stage. Several important experimental variables were optimized. The resulting biosensor showed a linear response to H2O2 over a concentration range from 4 to 100 mu M, with a sensitivity of 12.8 nA/mu M cm(-2) and a detection limit of 1.6 mu M, calculated as (3 S.D./sensitivity). The apparent Michaelis-Menten constant K-III(app) was calculated to be 0.295 +/- 0.020 mM. The biosensor showed high sensitivity as well as good stability and reproducibility. The performance of the biosensor was evaluated with respect to four possible interferences. (C) 2008 Elsevier Ltd. All rights reserved.