A genetically encoded fluorescent indicator was developed for the detection and characterization of estrogen agonists and antagonists. Two different color mutants of green fluorescent protein were joined by a tandem fusion domain composed of LXXLL (L = leucine, X = any amino acid) motif from the nuclear receptor-box II of steroid receptor coactivator 1, a flexible linker sequence, and the estrogen receptor alpha ligand binding domain (ERalpha LBD). Monitoring real-time ligand-induced conformational change in the ERalpha LBD to recruit the LXXLL motif allowed screening of natural and synthetic estrogens in single living cells using fluorescence resonance energy-transfer technique. The indicator was named SCCoR (single cell-coactivator recruitment). The high sensitivity of the present indicator made it possible to distinguish between estrogen strong and weak agonists in a dose-dependent fashion, immediately after adding ligand to live cells. Discrimination of agonists from antagonists was efficiently achieved using the present study. The approach described here can be applied to develop biosensors for other hormone receptors as well.