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In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is − 1.697 kcal/mol and ΔG = − 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.