Aminoacylase (EC 3.5.1.14) was immobilized via covalent bonding to magnetic iron oxide (Fe3O4) nanoparticles with 3-(aminopropyl)triethoxysilane (APTES) modification. Magnetic Fe3O4 nanoparticles were prepared by chemical co-precipitation, controlling n(Fe3+):n(Fe2+) = 2:1 and c(Fe3+) + c(Fe2+) = 0.3 mol/L. The nanoparticles modified with APTES were characterized by SEM and FT-IR. The results showed that the optimal nanoparticle concentration, glutaraldehyde concentrations, crosslink time and immobilization time are 8 mg/mL, 1.0%, 1 h, and 1.5 h, respectively. Moreover, the optimal temperature, pH and thermostability of free and immobilized enzymes were compared. The properties of repeatedly used immobilized aminoacylase were also investigated. (C) 2015 Elsevier B.V. All rights reserved.