A biosensor based on catalase enzyme was developed for the investigation of the effect of calcium ions on the activity of the enzyme. Calcium plays an activator role for the catalase enzyme that catalyses the degradation of hydrogen peroxide to 02 and H(2)O. Determination method of the effect of calcium ion on the activity of the enzyme was based on the assay of the differences on the responses of the biosensor in the absence and the presence of calcium in the reaction medium. The biosensor had a linear relation to calcium concentrations and good measurement correlation between 1 and 10 mM with 1 min response time. Tris-HCl buffer (pH 7.0; 50 mM) and 37 degrees C were obtained as the optimum working conditions. In the application studies, the biosensor was used determination of calcium level of real samples such as milk, spring and mineral water. (c) 2008 Elsevier Ltd. All rights reserved.