We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-NC) or alkaline treatment (OH-NC) of microcrystalline cellulose, which was subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) β-galactosidases. The mean size of magnetic nanocellulose particles was approximately 75 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with a coercivity of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose either in permeate or in cheese whey for 30× and it reached hydrolysis higher than 50%. Furthermore, using a continuous process in a column packed-bed reactor, the derivative OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or whey after 24 h of reaction. Fungal β-galactosidases immobilized on magnetic nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.