This study focuses on the immobilization, optimization, and characterization of protease enzyme secreted by the endophytic fungus Alternaria alternata. Proteases are crucial enzymes with wide applications in industrial processes, including food, pharmaceuticals, and biotechnology. To enhance the stability and reusability of these enzymes, immobilization techniques were employed. The immobilization of protease enzyme using chitosan-PVA matrix has been demonstrated to be effective in enhancing their stability, activity and making it a promising approach for various biotechnological applications. The optimization of immobilized proteases such as pH, thermal stability, storage stability and reusability were carried out using statistical design of experiments to maximize enzyme yield and activity. Characterization of the immobilized protease including FTIR and FE-SEM analysis was performed, which provided valuable information about the immobilization process, the structure and activity of the immobilized enzyme and the interaction between the enzyme and chitosan-PVA matrix. The immobilized protease exhibited improved thermal stability and retained significant activity after multiple cycles of use, demonstrating its potential for industrial applications. This research provides valuable insights into the efficient immobilization and application of fungal proteases, contributing to the development of sustainable and cost-effective biocatalytic processes.