The isolation and preconcentration of estrogens from new types of biological samples (acellular and protein-free simulated body fluid) by molecularly imprinted solid-phase extraction has been described. In this technique, supramolecular receptors, namely molecularly imprinted polymers (MIPs) are used as a sorbent material. The recognition sites of MIPs were prepared by non-covalent multiple interactions and formed with the target 17beta-estradiol as a template molecule. High-performance liquid chromatography with spectroscopic UV, selective, and a sensitive electrochemical CoulArray detector was used for the determination of 17beta-estradiol, estrone, and estriol in simulated body fluid which mimicked human plasma.