Estrogen receptor α/β (ERα/β), are members of the steroid/nuclear receptor superfamily. They function as a ligand-inducible transcriptional regulator and are targets for the treatment of several endocrine diseases. Irrespective of the ligand binding status, ERα/β are primarily localized in the nucleus. However, when activated, they bind to specific DNA response elements as homo-/hetero-dimers to transactivate their target genes. Homo-/hetero-dimerization of ERα/β is crucial in ER-mediated regulation of gene expression and cellular responses. However, the cellular site that hosts the receptor homo-/hetero-dimerization, essential for its physiological function, is poorly explored. Here, using a comprehensive approach, we show that the initial intermolecular interaction between ERα/β monomers occurs in the cytoplasmic compartment of the cell in a ligand-independent manner. To explore the site of homo-/hetero-dimerization of ERα/β in living cells, we created GFP-ERα/β and mCherry-ERα constructs to perform co-receptor interaction studies through fluorescence microscopy and live-cell imaging. The study revealed that ERα/β monomers dimerize in the cytoplasmic compartment, facilitating the nuclear import of the complex. We also observed that ligand-independent homodimerization requires a functional nuclear localization signal in at least one of the ER monomers. Finally, it has also been shown that the ligand-binding domain of ERα plays a key role in ligand-independent homodimerization. Understanding the intricacies of ER homo-/hetero-dimerization events and their disease-associated dysregulation paves the way for potential therapeutic interventions.