The mechanism of interaction between C.I. Direct Yellow 9 and human serum albumin was studied using spectroscopic methods including fluorescence spectra, UV–vis, Fourier transform infrared (FT-IR) and circular dichroism (CD). The quenching mechanism was investigated in terms of the association constants, number of binding sites and basic thermodynamic parameters. The distance between the human serum albumin donor and the acceptor dye was 3.64 nm as derived from fluorescence resonance energy transfer. Alteration of the secondary protein structure in the presence of the dye was confirmed by UV, FT-IR and CD spectroscopy. Molecular modeling revealed that a dye–protein complex was stabilized by hydrophobic forces and hydrogen bonding, via amino acid residues.