In this presented work, magnetic poly(2-hydroxyethyl methacrylate) (p (HEMA)) nanoparticles were synthesized by surfactant-free emulsion polymerization technique. Cibacron Blue F3GA was covalently attached to the magnetic p (HEMA) nanoparticles and Cu ions were then chelated with dye molecules. Synthesized magnetic nanoparticles were spherical with the diameter of 80 nm and exhibited magnetic character. Incorporation rate of Cibacron Blue for magnetic nanoparticles was found to be 28.125-μmol/g polymer. Loaded amount of Cu ions was calculated as 10.229-μmol/g polymer. These Cu-Cibacron Blue F3GA-derived magnetic p (HEMA) nanoparticles were used for urokinase adsorption under different conditions (i.e., pH, enzyme initial concentration, ionic strength, temperature). Maximum adsorption capacity was found to be 630.43-mg/g polymer, and it was observed that Langmuir adsorption isotherm was applicable in this adsorption process. The adsorbed urokinase was desorbed from the Cu-Cibacron Blue F3GA-derived magnetic p (HEMA) nanoparticles by using 1.0 M of NaCl with the desorption rate of 96%. It was also demonstrated that adsorption capacity did not change significantly after five adsorption/desorption cycles.