Novel double yolk–shell structure molecularly imprinted polymers were synthesized by surface polymerization with 17β-estradiol as the template, followed by chemical etching. The prepared polymers were characterized by transmission electron microscopy, nitrogen adsorption–desorption experiments, thermal gravimetric analysis and magnetic property experiments. For 17β-estradiol, the double yolk–shell structure showed a 60% improvement in the binding capacity compared to the single layer structure, and the cycle life was at least 18 times. The obtained polymers were applied as the adsorbent for magnetic solid-phase extraction (MSPE) for 17β-estradiol. Under the optimal conditions, an MSPE-HPLC method was developed for the detection of 17β-estradiol in milk powder. A wide linearity range of 0.3–300 μg kg−1 (R2 = 0.9997) for 17β-estradiol was obtained, and the limit of detection was 0.1 μg kg−1. Satisfactory recoveries ranged from 92.50% to 101.1% and relative standard deviations were lower than 6.1%. The established MSPE-HPLC method has been proven to be accurate for the analysis of 17β-estradiol in milk powder.
