The effects of different operational conditions of α-amylase covalent immobilization on magnetic nanoparticles (MNPs), such as initial enzyme concentration, glutaraldehyde (GA) concentration, pH, and ionic strength were investigated using a central composite design (CCD). Moreover, two responses, the biocatalyst activity and amount of immobilized enzyme were simultaneously studied using Derringer’s desirability function. The optimum amount and activity of immobilized enzyme were determined to be 24.83% and 556.41 mg g_MNP⁻¹ at an initial enzyme concentration of 999.86 ppm, solution pH of 4.6, GA concentration of 0.59%, ionic strength of 99.99 mM and a process time of 4 h. The study of the kinetic parameters and enzyme stability showed a significant enhancement in the performance of the immobilized enzyme with respect to the free enzyme. The storage stability and reusability of the immobilized biocatalyst were found to be about 50 and 40% of the initial activity after 12 days and 6 cycles, respectively.