Magnetic nanobiocatalysts for tag cleavage on fusion proteins have been prepared by immobilizing enterokinase (EK) onto iron oxide magnetic nanoparticles coated with biopolymers. Two different chemistries have been explored for the covalent coupling of EK, namely carbodiimide (EDC coupling) and maleimide activation (Sulfo coupling). Upon immobilization, EK initial activity lowered but EDC coupling lead to higher activity retention. Regarding the stability of the nanobiocatalysts, these were recycled up to ten times with the greater activity losses observed in the first two cycles. The immobilized EK also proved to cleave a control fusion protein and to greatly simplify the separation of the enzyme from the reaction mixture.