Magnetic functionalized chitosan-based nanocarrier proffered to purify protein and enzyme has incepted a striking place in separation and purification predominantly for its convenient and rapid performance. Here, the surface modification of chitosan biopolymer (CS) was employed by terephthaloyl chloride, dopamine (DA), and NiCl₂ (Ni²⁺), followed by an in-situ magnetization by Fe₃O₄ nanoparticles (NPs). This magnetic nanocomposite was utilized for alkaline phosphatase separation from bovine milk. Based on the FE-SEM images, the particle size narrow range between 55.4 nm and 72.1 nm was measured, and the spherical NPs represented a mean diameter of 66.9 nm. Moreover, the reduction in the magnetization saturation (MS) of synthesized CS-DA/Ni²⁺/Fe₃O₄ nanocomposite (3.18 emu/g) compared to Fe₃O₄ MNPs’ MS (44.2 emu/g) is correlated to the addition of non-magnetic shells. The activity measurements of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase (ALPs) were administrated for purification process investigation. Considering the purification yield of the extracted protein concentration by mentioned nanocomposite, the most efficient ALPs protein purification was about 67% after 30 min incubation at 30 °C through a cost-efficient and reproducible approach. Due to the affinity purification, the Ni²⁺ affinity ligand immobilized on the nanocarrier presented high stability. After five cycles, a very low adsorption capacity reduction was detected, and 94% of the original binding capacity of the magnetic nanocomposite was retained, indicating high reusability of the nanocomposite and the specified affinity ligand selection toward ALPs.