The novel magnetic molecularly imprinted polymers (MIPs) were prepared by using 17-β-Estradiol as template molecule, methacrylic acid as functional monomer, trimethylol propyl triacrylate as crosslinker, dithio acid (isobutyronitrile) ester as initiator, and Fe3O4 as magnetic fluid, for detection of 17-β-Estradiol using 7-hydroxy evil diazoxide ketone as fluorescent probe.Selective adsorption of the magnetic MIPs was studied with selection coefficient of 115.72 and 36.42 for the estradiol and bisphenol A, respectively.The maximum adsorption capacity for estradiol was obtained of 280.99 μmol/g.Excellent linearity toward 17-β-Estradiol was attained within 0.05-18 nmol/mL (R=0.986) and the limit of detection was 0.0261 nmol/mL.The magnetic imprinting microspheres were demonstrated great potential for highly selective and sensitive fluorescent probing of trace 17-β-Estradiol based on its competitively substituting the 7-hydroxy evil diazoxide ketone.