A novel nanosized substrate imprinted polymer (MIPs-GO-Fe3O4) was developed on a magnetic graphene oxide (GO-Fe3O4) surface for selective recognition and fast removal of 17β-estradiol (17β-E2). The characteristics of MIPs-GO-Fe3O4 were evaluated by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy. and vibrating sample magnetometer (VSM). Results suggested that GO had a thin single-layer structure anchoring Fe3O4 nanoparticles and that the imprinted film was coated on the GO-Fe3O4 surface. MIPs-GO-Fe3O4 was sensitive to the magnetic field and could be easily separated using an external magnet. The adsorption results indicated that the kinetic value and binding capacity of MIPs-GO-Fe3O4 were 0.0062 g (mg·min)(-1) and 4.378 μmol g(-1), respectively. The Langmuir-Freundlich isotherm and pseudo-second-order kinetic models were the main adsorption mechanisms for MIPs-GO-Fe3O4. MIPs-GO-Fe3O4 showed excellent recognition selectivity, as well as enrichment and separation abilities for 17β-E2 in complex matrices. MIPs-GO-Fe3O4 was also used to analyze 17β-E2 in real food samples, and satisfactory recoveries such as 84.20% with relative standard deviation (RSD) of 4.67% at a spike of 0.5 μmol L(-1) were obtained. Thus, the MIPs-GO-Fe3O4-based method provided a convenient and practical platform for the separation, enrichment, and removal of 17β-E2 in food samples.