Background Sodium fluorescein (SF) is a fluorescent tracer dye used extensively in diagnostic tools in the field of Ophthalmology, particularly in intravenous fluorescein angiography (IVFA). The binding of SF to human serum albumin (HSA) has been predicted by molecular docking and investigated by circular dichroism (CD) and fluorescence spectroscopy with or without glycation at temperatures 296, 301, and 310 0.25 K. Methods The binding parameters were calculated by quenching of emission spectrum of a constant concentration of SF (2 0.25 μmol/l) at 513 0.25 nm against increasing concentrations of glycated or unmodified HSA as quencher starting from stoichiometry ratio of 1:1. Results The HSA-SF interaction found to be a static binding. The Stern-Volmer constants (Ksv) were in the range of ~ 0.10 104 0.25 M− 0.10 1 and other thermodynamic parameters like enthalpy (ΔH°), free energy (ΔG°) and entropy (ΔS°) are similar to albumin ligand bindings reported by previous workers. Conclusions The interactions were found to be spontaneous, irrespective of temperature or glycation. Glycated HSA is clinically used to monitor unstable glycemic controls in diabetic patients. A 39% increase in binding affinity (log K) and free energy (ΔG°) is reported on glycation at 310 0.25 K (room temperature), which may be important in the SF based angiographies. On glycation HSA-SF binding appears to change from an enthalpy-driven to an entropy-driven reaction. SF shows best binding to FA binding site III of HSA, which also overlaps with drug binding site II of subdomain IIIA. Leu430 seems to play a pivotal role in the interaction. © 2017