A novel 17-estradiol molecularly imprinted polymer was grafted onto the surface of initiator-immobilized silica by surface-initiated atom transfer radical polymerization. The resulting molecularly imprinted polymer was characterized by elemental analysis and thermogravimetric analysis. The binding property of molecularly imprinted polymer for 17-estradiol was also studied with both static and dynamic methods. The results showed that the molecularly imprinted polymer possessed excellent recognition capacity for 17-estradiol (180.65 mg/g at 298 K), and also exhibited outstanding selectivity for 17-estradiol over the other competitive compounds (such as testosterone and progesterone). Then, the determination of trace 17-estradiol in beef samples was successfully developed by using molecularly imprinted polymer solid-phase extraction coupled with high-performance liquid chromatography. The limit of detection was 0.25 ng/mL, and the amount of 17-estradiol in beef samples was detected at 2.83 ng/g. This work proposed a sensitive, rapid, reliable, and convenient approach for the determination of trace 17-estradiol in complicated beef samples.