Cellulose nanofibers (CNFs) are one of attractive supporting materials for enzyme immobilization due to their unique properties such as high surface area, high porosity and surface carboxyl groups for chemical bonding. In this study, CNFs were prepared via TEMPO-mediated oxidation and physical grinding of cellulose, and further used for the immobilization of alpha-chymotrypsin (CT) enzyme via four different approaches such as covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically-separable EPC (Mag-EPC). EPC approach consists of three steps: covalent enzyme attachment, enzyme precipitation and crosslinldng, while EC represents a control without the step of enzyme precipitation. Amine-functionalized magnetic nanoparticles were added during the enzyme precipitation and crosslinking steps to produce magnetically-separable EPC. The activities of CA, EC, EPC and Mag-EPC were 0.067, 0.14, 1.3 and 2.6 units per mg CNFs, respectively, representing that the activity of Mag-EPC was 38-, 19- and 2-times higher than those of CA, EC and EPC, respectively. After incubation under shaking (200 rpm) for 30 days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after 8 days. Because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis, the present results of stable EPC and Mag-EPC on CNFs have demonstrated the great potential of CNFs as an environmentally-friendly and economical carrier of enzyme immobilization, which allows for magnetic separation as well as high enzyme activity/loading and stability. (C) 2017 Elsevier B.V. All rights reserved.