A new amperometric malate enzyme electrode probe has been constructed using a hydrogen peroxide-based sensor coupled with malic and pyruvate oxidase enzymes. The first enzyme catalyzes the oxidation of malic acid, which in the presence of NADP+ yields pyruvate as product. The oxidation of pyruvate is catalyzed by pyruvate oxidase, which yields H2O2 as product in the presence of O2 and phosphate as cosubstrates and thiamine pyrophosphate and Mg2+ as cofactors. The H2O2 is then detected by the electrochemical transducer, and the output current changes are correlated to the concentration of malic acid in solution. Analytical parameters such as pH, temperature, buffer, substrate and cofactor concentrations, and response time have been optimized. Probe stability and reproducibility have been evaluated. The malic enzyme was used first in solution and then coimmobilized with pyruvate oxidase. Coimmobilization of the oxidase and dehydrogenase enzymes has been performed both randomly and asymmetrically on differen...