A novel biosensor for homocysteine determination has been developed. The biosensor was fabricated with L-homocysteine clesulfhydrase immobilized on the ammonium selective electrode by means of eggshell membrane. The measurement principle is based on determination of ammonia due to the enzymatic reaction in the medium by ammonium selective electrode. The effects of enzyme loading, glutaraldehyde concentration, pH, buffer concentration, temperature, dithiotreitol (DTT) concentration and ionic strength adjustment buffer (ISA) on the biosensor response were investigated in detail. The linear detection range and limit of detection (LOD) for homocysteine were found to be 0.15 - 1.8 mM and 55 mu M, respectively. Finally, the homocysteine biosensor has been applied to plasma samples for determination of total homocysteine contents. (c) 2007 Elsevier B.V. All rights reserved.