A molecularly imprinted polymer (MIP) has been synthesized by a thermo-polymerization method using methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as cross-linker, acetonitrile as porogenic solvent, and 17beta-estradiol as template. The MIP showed obvious affinity for 17beta-estradiol in acetonitrile solution, which was confirmed by absorption experiments. After optimization of molecularly imprinted solid-phase extraction (MISPE) conditions, three structurally related estrogenic compounds (17beta-estradiol, estriol, and diethylstilbestrol) were used to evaluate the selectivity of the MIP cartridges. The MIP cartridges exhibited highly selectivity for E(2), the recoveries were 84.8+/-6.53% for MIPs and 19.1+/-1.93% for non-imprinted polymer (NIP) cartridges. The detection and quantification limits correspond to 0.023 and 0.076 mg L(-1). Furthermore, the MISPE methods were used to selectively extract E(2) from fish and prawn tissue prior to HPLC analysis. This MISPE-HPLC procedure could eliminate all matrix interference simultaneously and had good recoveries (78.3-84.5%).