Raman confocal microscopy with 488 nm excitation wavelength supported with atomic force microscopy (AFM), scanning near-field optical microscopy (SNOM) and UV–Vis spectrometry was used to investigate air-dried erythrocytes (red blood cells, RBCs) in whole human blood smears. The central internal part of the cell was dominated by the laser-induced O2 dissociated oxyHb form as evidenced by the Fe2+ marker band appearing at 1356 cm−1. The existence of a thin outer layer of hemoglobin in the periphery of RBCs was assigned to hemichrome. Evidence for hemichrome includes the oxidation state marker band appearing at 1376 cm−1, the absence of FeO2 band at 570 cm−1 and a UV–Vis spectrum consistent with hemichrome. This is the first time that distributions of Fe2+/Fe3+ hemes inside the single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leucocytes and carotenoids of blood plasma.