Estrogen Receptor (ERα) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERα for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERα modulator. We cloned the ERα LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Erα(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERα(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERα modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 mmol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 mmol/L. Using this screening model, we discovered four ERα agonists from 2000 natural and synthetic compounds.