The immobilization of cellulase on amine-functionalized FeO magnetic nanoparticles (MNPs), via metal affinity immobilization, as a nano-biocatalyst was investigated. Copper was chosen as ligand and loaded onto MNPs in a buffering environment without adding any intermediates. Immobilization conditions were optimized by a 2 full factorial design method. Under optimized working conditions (Cu/MNPs = 1, E/MNPs = 0.11, pH = 6), the relative enzyme activity and the amount of enzyme immobilization were 91% and 164 (mg enzyme/g MNPs), respectively. The immobilized cellulase (tested by carboxymethyl cellulose hydrolysis at 1% concentration) was found to be more stable than the free enzyme. Also, the immobilized enzyme still retained 73% of its initial activity after five cycles of usage. Furthermore, the free and immobilized cellulases retained 70 and 84% of their initial activity after eight days storage at 4 °C, respectively. Immobilization of enzymes, using this method, could be a good and economic option for various industries.