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Here, we report the glycosylation of human adult hemoglobin (Hb) studied in aqueous solution and at the air–water interface by the Langmuir–Blodgett (LB) technique. Pressure–area (π–A) and pressure–time (π–t) measurements show that the concentration of glucose (GLC) and interaction time have an effect on Hb molecular area as well as on surface activity. Solution studies by UV-vis absorption and emission spectroscopy show that the GLC can alter the local conformation of Hb to some extent at the tryptophan and heme residues. CD spectroscopic studies in solution indicate that the α-helix content increases in the presence of GLC at the secondary structure level, which may be the cause of an increased adsorption rate of Hb. Also, secondary structure calculation using FTIR technique in the LB film follows the decrease in α-helix and increase in β-sheet structure as well as the formation of intermolecular …