Abstract: To achieve efficient immobilization of papain (PAP), this study constructed a hydrophilic three-dimensional matrix for easy magnetic separation of the immobilized enzyme. The immobilization conditions were optimized using single-factor and response methodology. A core-shell structured nano-magnetic bead Fe₃O₄@PMMA was prepared by coating the magnetic Fe₃O₄ with poly(methyl methacrylate) (PMMA), followed by grafting polyethyleneimine (PEI) of different molecular weights. PAP was physically adsorbed onto the PEI, and then cross-linked using glutaraldehyde (GA) to construct an easily magnetically separable hydrophilic three-dimensional immobilization matrix Fe₃O₄@PMMA-PEI. The cross-linked encapsulation of PAP and its microstructure and physicochemical properties were characterized. Using the immobilization capacity of the nano-magnetic beads as the evaluation criterion, the optimal cross-linking time, glutaraldehyde concentration, cross-linking temperature, and cross-linking speed for immobilizing PAP were investigated. The results indicated that the carboxyl group content formed after the ester hydrolysis of the PMMA on the surface of the magnetic beads was 0.95 mmol/g. Following the grafting of PEI with different molecular weights, the amino group content on the surface of the magnetic beads was 0.16~0.48 mmol/g. The loading capacity of PAP on the nano-magnetic beads could be modulated by the molecular weight of the grafted PEI. When the molecular weight of PEI was 1800, the loading capacity reached 120.71 mg/g. PEI 1800 was selected as the grafting spacer arms. The nanomagnetic spheres exhibited well-defined morphology, uniform particle size distribution in the range of approximately 150~200 nm. Through response surface experiments, the best conditions were optimized: Cross-linking time of 32 minutes, glutaraldehyde concentration of 0.02%, cross-linking temperature of 25 ℃, and cross-linking speed of 90 revolutions per minute. Under these conditions, Fe₃O₄@PMMA-PEI 1800 achieved a PAP loading capacity of 139.80 mg/g. Importantly, even after five cycles of reuse, Fe₃O₄@PMMA-PEI 1800-PAP retained 80.35% of its relative enzyme activity. This study presents the design and synthesis of a nanomagnetic sphere structure tailored for the efficient immobilization of PAP, providing experimental evidence for the research and application of immobilized PAP.