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Estrogen receptors (ERs), comprising ER α and ER β, are crucial for regulating cell growth and differentiation via homo- and hetero-dimer formation. However, accurately detecting ER dimerization with precise spatiotemporal resolution remains a significant challenge. In this study, fluorescence resonance energy transfer-based biosensors to monitor ER dynamics in real-time, are developed and optimized. This approach involves comprehensive structural analysis, linker comparison, and the selection of optimal fluorescent protein pairs, resulting in three distinct biosensors capable of detecting all ER homo- and hetero-dimerizations within the nucleus. These biosensors are utilized to reveal interactions between ER α/β and calmodulin during dimer formation. Furthermore, by leveraging the ligand-binding domain (LBD) of ER β, ER ββ LBD biosensor is designed for real-time analysis of ER ββ homodimerization in the cytoplasm, enhancing the ability to screen ER dimerization-related drugs. Additionally, we developed a novel ER ββ translocation biosensor, which enables real-time observation of ER ββ translocation to the nucleus-a capability previously unavailable, is developed. This spatiotemporal analysis demonstrates the relevance of ER translocation in response to drug binding efficacy and extracellular matrix changes. Our biosensors offer transformative tools for studying ER dynamics, providing valuable insights for drug screening and the investigation of ER-related cellular processes., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)