Alpha-amylases belong to the endoamylase family, which perform the primary hydrolysis of starch to smaller oligosaccharides by breaking the (4→1)α bond. Among the different types of enzymes, alpha-amylases constitute an important group with wide applications in various industries. The use of free enzymes in various industries has limitations, including: high cost, lower stability, poor reusability, and difficulty in using them in continuous reactors. These limitations can be minimized by immobilizing the enzyme. Enzyme immobilization can be performed on various substrates. In the meantime, enzyme immobilization on nano-surfaces, especially magnetic nanoparticles, has received much attention due to their low toxicity, reusability of the enzyme along with increased stability and robust performance under various conditions. In this study, alpha-amylase enzyme immobilization was performed using Fe2O3 nanoparticles on a chitosan polymer substrate. For the initial production of the enzyme, an overnight culture of Bacillus mujavensis was inoculated into a specific liquid medium and incubated for 48 hours. The obtained enzyme was concentrated using ammonium sulfate and then dialyzed. The dialyzed enzyme was bound to Fe2O3 nanoparticles in a chitosan substrate in the presence of 50% glutaraldehyde (binding agent). The activity and stability of the enzyme immobilized with nanoparticles were measured at 540 nm and were found to be 53 and 48% higher than the free enzyme, respectively. The concentration of the immobilized enzyme was also estimated using a standard curve of 25 μg bovine serum albumin.