As endocrine-disrupting chemicals, a few frequently used phthalate plasticizers were banned or restricted for use as additives in food in some countries. The interaction mechanisms between three phthalate plasticizers with human serum albumin (HSA) were studied by fluorescence (quenching, synchronous, and three-dimensional), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, in combination with molecular modeling under simulative physiological conditions, respectively. The results obtained from fluorescence quenching data revealed that the plasticizers-HSA interaction altered the conformational strcture of HSA. Meanwhile, the alterations of HSA secondary structure in the presence of phthalate plasticizers were investigated. The binding distances for the plasticizers-HSA system were provided by the efficiency of fluorescence resonance energy transfer. Furthermore, the thermodynamic analysis implied that hydrophobic forces were the main interaction for the plasticizers-HSA system, which agreed well with the results from the molecular modeling study.