The stability of enzyme-conjugated magnetic iron oxide nanoparticles in plasma is of great importance for in vivo delivery of the conjugated enzyme. In this study, β-glucosidase was conjugated on aminated magnetic iron oxide nanoparticles using the glutaraldehyde method (β-Glu-MNP), and further PEGylated via N-hydroxysuccinimide chemistry. The PEG-modified, β-glucosidase-immobilized magnetic iron oxide nanoparticles (PEG-β-Glu-MNPs) were characterized by hydrodynamic diameter distribution, zeta potential, Fourier transform infrared spectroscopy, transmission electron microscopy, and a superconducting quantum interference device. The results showed that the multidomain structure and magnetization properties of these nanoparticles were conserved well throughout the synthesis steps, with an expected diameter increase and zeta potential shifts. The Michaelis constant was calculated to evaluate the activity of conjugated β-glucosidase on the magnetic iron oxide nanoparticles, indicating 73.0% and 65.4% of enzyme activity remaining for β-Glu-MNP and PEG-β-Glu-MNP, respectively. Both magnetophoretic mobility analysis and pharmacokinetics showed improved in vitro/in vivo stability of PEG-β-Glu-MNP compared with β-Glu-MNP. In vivo magnetic targeting of PEG-β-Glu-MNP was confirmed by magnetic resonance imaging and electron spin resonance analysis in a mouse model of subcutaneous 9L-glioma. Satisfactory accumulation of PEG-β-Glu-MNP in tumor tissue was successfully achieved, with an iron content of 627±45 nmol Fe/g tissue and β-glucosidase activity of 32.2±8.0 mU/g tissue.