Glucocorticoid receptor (GR) is a hormone-activated transcription regulatory protein involved in metabolism as well as adrenocortical responses to psychosocial stress. Ligand-activated GR localizes to the nucleus, where GR homodimers regulate gene transcription via direct binding to glucocorticoid response elements (GREs). The role of GR homodimers in transcriptional activation has not yet been elucidated. In this study, we determined the concentration of GR homodimer, and its dissociation constant (Kd), at the single-cell level, by using fluorescence correlation spectroscopy (FCS) combined with a microwell system. Results from dissociation constant analysis and diffusion analysis suggested that GR forms complexes with other proteins as well as homodimers. We determined the relationship between the concentration of GR homodimer and transcriptional activity using a triple-color FCS-microwell system-based fluorescent reporter assay. The binding affinity of GR to GREs was analyzed via fluorescence cross-correlation spectroscopy (FCCS). Our findings indicate that the GR homodimer is essential for activating target gene transcription.