The food sector uses methyl yellow (MY) extensively as a colorant. The primary transporter in vivo that influences MY absorption, metabolism, distribution, and excretion is human serum albumin (HSA). Exploring the binding process and looking at how HSA and MY work physiologically at the molecular level is therefore very important. Experiments using steady-state fluorescence and fluorescence lifetimes proved that HSA and MY's quenching mechanisms were static. The HSA–MY complex's binding constant was estimated using thermodynamic parameters to be around 10⁴ M⁻¹. The hydrophobic forces were a major factor in the binding process, as evidenced by the negative ΔG, positive ΔH, and ΔS, which suggested that this contact was spontaneous. Site tests showed that MY linked to HSA's site I. Circular dichroism and three-dimensional fluorescence analysis revealed that the 1.33% α-helix content dropped and the amino acid microenvironment altered. While HSA's protein surface hydrophobicity decreased when engaging MY, the binding of MY to HSA reduced in the presence of urea. The stability of the system was assessed using molecular modeling. Additionally, HSA's esterase-like activity decreased when MY was present, and Ibf/Phz affected the inhibition mechanism of MY on HSA. These findings offer a distinctive perspective for comprehending the structure and functioning of HSA and evaluating the safety of MY.