In this study an amperometric biosensor based on pyruvate oxidase was developed for the determination of pyruvate and phosphate. For construction of the biosensor pyruvate oxidase was immobilized with gelatin and insolubilized in film by forming cross-linked bonds with glutaraldehyde. The film was fixed on a YSI type dissolved oxygen (DO) probe, covered with a teflon membrane which is high-sensitive for oxygen. The working principle of the biosensor depends on detection of consumed DO concentration related to pyruvate concentration which is used in enzymatic reaction catalyzed by pyruvate oxidase. The biosensor response shows a linearity with pyruvate concentration between 0.0025 and 0.05 mu M and also response time of the biosensor is 3 min. In the optimization studies of the biosensor the most suitable enzyme activity was found as 2.5 U/cm(2) for pyruvate oxidase, and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were established as providing the optimum working conditions. In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity, operational stability, determination of phosphate, and interference effects of some compounds on the pyruvate determination were investigated. Finally, the concentration of pyruvate was determined by using spectrophotometric method and the results obtained were compared to results obtained by the biosensor. (C) 2007 Elsevier Ltd. All rights reserved.