Intrinsically disordered proteins (IDPs) or regions (IDRs) are thought to exhibit unique functionalities without forming ordered structures. However, these molecular mechanisms are not easily elucidated, partly because of the difficultly of measuring structural information. In this study, we applied the alternative laser excitation (ALEX) method and circular dichroism (CD) spectroscopy to investigate the structure of the C-terminal tail (CTT) domain of the human epidermal growth factor receptor (EGFR). The single-molecule distributions of Forster resonance energy transfer (FRET) obtained by ALEX under solution conditions modified by the addition of potassium chloride (KCI), urea, or guanidinium chloride (GdmCl) allowed us to separately examine the influences of charge interactions and secondary structure formation. The CD spectrum analyses indicated the types of included secondary structure. The results suggested that the structure of the CTT is influenced by secondary structure formation, which is a principally antiparallel beta-sheet, rather than by charge interactions and that phosphorylation of the major Grb2-binding sites partially denatures that secondary structure. Our findings suggest that the EGFR CTT might regulate ligand binding kinetics by local beta-sheet formation or by the disruption associated with phosphorylation states.