A simple and low-cost hydrogen peroxide biosensor was fabricated using a beef liver catalase-immobilized eggshell membrane and an oxygen electrode. Catalase from beef liver extract was covalently immobilized on an eggshell membrane and subsequently covered the surface of an oxygen electrode. The detection scheme was based on the increase in dissolved oxygen content upon exposure to hydrogen peroxide solution. The increase in oxygen level was then monitored and related to the hydrogen peroxide concentration. The effects of enzyme loading, dissolved oxygen content, pH, phosphate buffer concentration and temperature on the biosensor have been studied in detail. The response and recovery times of the biosensor (t(95)) were 1 min, respectively. The detection limit based on 3sigma(b) was 3 muM and the relative standard deviation of the response was 1.28% (n=10) for a solution containing 0.3 mM of H2O2. The hydrogen peroxide biosensor demonstrated a reasonable long and stable shelf-life. It has been successfully applied to the determination of hydrogen peroxide concentration in some commercial samples. (C) 2003 Elsevier Inc. All rights reserved.