With growing interests of phytoestrogens, many natural phytochemicals extracted from diverse plant species have been explored for their estrogenic-like activities and potential applications. In this work, a simple and rapid separation of phytoestrogenic compounds from crude plant extracts was purposed using magnetic nanoparticles (MNPs) of Fe3O4 immobilized with the ligand-binding domain (LBD) of estrogen receptor alpha (ER alpha). The recombinant LBD-ER alpha peptide of 40 kDa was produced and subsequently covalently linked to MNPs. One milligram of the LBD-ER alpha-immobilized MNPs demonstrated a specific binding to the standard 17 beta-estradiol (E2) of 3.37 nmol and 91.3-100 % of the bound E2 were subsequently recovered. LBD-ER alpha-immobilized MNPs could separate phytoestrogens of 4.6 nmol E2-equivalent activity from a 1-mg crude extract of Asparagus racemosus. The produced MNPs showed no separation yield when were applied to the negative controls, the crude extract of radish (Raphanus sativus), and the standard progesterone (P4). Thin-layer chromatography demonstrated a single phytochemical band of the separated phytoestrogens, which exhibited the activity to promote MCF-7 cell proliferation at 4.7 folds greater than the crude A. racemosus extract. The results of this work demonstrated a simple method to specifically separate phytoestrogens from crude plant extracts via the LBD-ER alpha-immobilized MNPs.