<p>A molecularly imprinted polymer was synthesized—in which NTAA (nanoparticle Fe₃O₄/SiO₂-tetraethyl silicate;3-triethoxysilylpropan-1-amine-acryloyl chloride) was used to serve as a connecting bridge to link the magnetic nanoparticles (MNPs) to monomer; a cross-linker agent was also applied to link polymer chains. Therefore, NTAA can result in molecularly imprinted polymer (MIP) at the presence of azobisisobutyronitrile (initiation), ethylene glycol dimethacrylate (as cross-linker agent), acryloyl chloride (second monomer), casein (analyte), and ethanol–water (50:50) as solvent. Because of fluorescence properties of alpha-casein, fluorescence spectroscopy can be exploited to detect the adsorption amounts of casein, and the detection limit of casein adoption was 0.5 μmol.L^–1 which refers to the minimum concentration of casein that can be reliably detected in a given sample. Effects of various effective factors in casein adsorption were studied and optimized among which concentration of crosslinking agent, analyte volume used in MIP synthesis, analyte concentration in separation step, extraction time, pH, and temperature can be mentioned. Optimized fluorescence emission was achieved at 339.5 nm using a standard solution of casein in aqueous sodium hydroxide solution as a solvent. The intermediates formed during the MIP synthesis reactions were also identified by FT-IR and TLC. Magnetic properties of the nanoparticles were assessed by VSM; while the surface and size of nanoparticles and MIP were evaluated by XRD. Moreover, the surface morphology of MIP and NIP was examined by SEM images. Nanoparticles and MIP offered proper fluorescence efficiency through an excellent yield (%93) of casein separation from bovine’s milk.</p> Graphical Abstract <p></p>