The mechanisms underlying the interaction between the two optical isomers of astaxanthin (AST) and bovine serum albumin (BSA) were investigated based on multiple spectroscopic, surface plasmon resonance, and molecular docking analyses. Both AST isomers were found to bind to BSA at the junction of subdomains IIA and IIIA, in the absence of any significant effects on protein conformation. The two isomers had similar binding affinities [4.17×10-7 mol/L for (3S,3’S)-AST and 3.91×10-7 mol/L for (3R,3’R)-AST for] and kinetic binding processes (slow binding, slow dissociation) for BSA. However, at higher concentrations (0.35~1.78 μmol/L), the highest binding response values obtained for (3S,3’S)-AST were higher than those for (3R,3’R)-AST. In addition, the change in enthalpy (ΔH) for the interactions between (3S, 3’S)-AST and (3R, 3’R)-AST and BSA were -175.09 and -149.42 kJ/mol, respectively, and the corresponding changes in entropy (ΔS) were -502.72 and -417.65 J/(mol•K). The negative values obtained for ΔH and ΔS indicate that hydrogen bonds and van der Waals forces were the main forces underlying the AST-BSA interactions. Molecular docking analysis revealed that (3S,3’S)-AST formed 2.0 Å and 2.7 Å hydrogen bonds with the Lys504 and Thr190 residues in BSA, respectively, whereas (3R,3’R)-AST formed a 2.9 Å hydrogen bond with the Arg435 residue. Our findings in this study contribute to elucidating the mechanisms associated with the binding of AST optical isomers to BSA, and will provide important theoretical guidance for the potential pharmacokinetics of AST isomers in blood circulation.