Understanding estrogen receptor (ER) signaling pathways is crucial for uncovering the mechanisms behind estrogen-related diseases, such as breast cancer, and addressing the effects of environmental estrogenic disruptors. Traditionally, ER signaling involves genomic events, including ligand binding, receptor dimerization, and transcriptional modulation within cellular nuclei. However, recent research have revealed ERs also participate in non-genomic signaling pathways, adding complexity to their functions. Researchers use advanced fluorescence-based techniques, leveraging fluorescent probes (FPb) to study ER dynamics in living cells, such as spatial distribution, expression kinetics, and functional activities. This review systematically examines the application of fluorescent probes in ER signaling research, covering the visualization of ER, ligand-receptor interactions, receptor dimerization, estrogen response elements (EREs)-mediated transcriptional activation, and G-protein-coupled estrogen receptor (GPER) signaling. Our aim is to provide researchers with valuable insights for employing FPb in their explorations of ER signaling.