In this study, catalase enzyme was immobilized by entrapping in alginate beads in the presence of gelatin. In the optimization studies of the bioactive layer immobilized some parameters such as enzyme amount, alginate, gelatin, and crosslinking agent glutaraldehyde amount were determined as 700 U/mL, 2.0%, 18 mg/mL, and 5.0%, respectively. Effects of pH and temperature on the immobilization were also investigated. In the characterization studies of the immobilized enzyme storage and thermal stability experiments were done. The immobilized enzyme was used for the decomposition of hydrogen peroxide in milk samples and also by using a. catalase biosensor prepared by the decomposition level of hydrogen peroxide was detected.