Rapid screening and evaluation of endocrine disruption chemicals including environmental estrogens (EEs) is crucial for environmental safety and public health. Conventional methods such as animal tests and cell assays are costly, time consuming, and hardly reproducible. In this work, a fluorescence biosensor mimicking the molecular interactions in the estrogen receptor (ER) signaling pathway was developed for the rapid evaluation of estrogenic activity of environmental chemicals. The key element of the biosensor is a dual-function DNA probe composed of an ER binding sequence and a dye-binding sequence. The ER binding sequence is part of the estrogen response element in the ER signaling pathway and used to bind the activated ER. The dye-binding sequence consists of six thymine bases which the OliGreen fluorescent dye binds to selectively and thus labels non-covalently. In the presence of an estrogenic chemical, ER is activated and then complexed with the DNA, leading to a reduction in the fluorescence anisotropy of OliGreen. Detection of estradiol produced a dose-response curve with an EC