Molecular orientation of chloroperoxidase (CPO) on surface of Fe3O4 magnetic nanoparticles (MNPs) was achieved through layer-by-layer (LBL) controlled assembly by site specific interactions of avidin and biotin. Enzymatic oxidative decolorization of soluble aniline blue was employed to evaluate the catalytic performance of immobilized CPO (I-CPU). The decolorization efficiency of dye by I-CPO was higher than 90% within 10 min. Moreover, I-CPO displayed improved thermostability and kept higher activity in wider pH range and in the presence of high concentration of oxidant (H2O2) compared to that of free enzyme. When incubated at 70 degrees C for an hour, 42.35% of the activity of immobilized enzyme was reserved compared with that at 25 degrees C, whereas free CPO remained only 8.46%. The tolerance to H2O2 was improved from 2 mmol L-1 to 4 mmol L-1, and the suitable pH range become wider. The relative activities of I-CPO in the 20 times use maintained 62% of that in the first run. The enzymatic kinetic constants confirmed that the entrapping CPO into this well-defined architecture could reduce diffusional limitation of reactants and products. (C) 2014 Elsevier B.V. All rights reserved.