The interaction between human serum albumin (HSA) and arachidonic acid (AA) as an unsaturated fatty acid were investigated in the present study using methods including UV-VIS spectrophotometry, fluorescence and circular dichroism (CD) spectroscopy, lifetime measurements, fluorescence anisotropy measurements and visual molecular dynamics (MD). The thermodynamic parameters were assessed from HSA thermal and chemical denaturation in the presence and absence of AA. From the thermal denaturation, the Tm and ΔG˚(298K) magnitudes obtained were 327.7 K and 88 kJ/mol, respectively, for HSA alone, and 323.4 K and 85 kJ/mol, respectively, following treatment with a 10 µM AA concentration. The same manner of reduction in Gibbs free energy as a criterion of protein stability was achieved during chemical denaturation by urea in the presence of AA. The present study investigates HSA binding nature through MD approaches, and the results indicated that the binding affinity of AA to the subdomain IIA of HSA is greater compared with that of subdomain IIIA. Although the HSA regular secondary structure evaluation by CD exhibited a minor change following incubation with AA, its tertiary structure revealed an observable fluctuation. Thus, it appears that the interaction between AA and HSA requires minor instability and partial structural changes.