A new amperometric biosensor based on urate oxidase-peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H2O2 from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 muM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 degreesC were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9 x 10(-3) U cm(-2) for urate oxidase and 512.7 U cm(-2) for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out. (C) 2003 Elsevier Science B.V. All rights reserved.