Perfluorododecanoic acid (PFDoA) is the most toxic emerging environmental contaminant among the 8～12 carbon chain perfluoroalkyl acids (PFAAs). A large amount of knowledge in the field of environmental PFAAs has been accumulated so far, while we are still just at the beginning of research into the interaction between PFDoA and human serum albumin (HSA). The goal of this study was to comprehensively determinate the binding mechanism of PFDoA with HSA by using fluorescence quenching technique in combination with molecular modeling and circular dichroism (CD) spectroscopy under the simulative physiological conditions. The quenching of HSA fluorescence by PFDoA was found to be a result of the combination of dynamic quenching and the formation of PFDoA-HSA complex. The calculated binding distance (r=3.65 nm) indicated that the non-radioactive energy transfer came into being in the interaction between PFDoA (acceptor) and HSA (donor). By performing displacement measurements, the specific binding of PFDoA in the vicinity of site I of HSA was clarified. Furthermore, the binding details between PFDoA and HSA were further confirmed by molecular docking studies, which revealed that PFDoA was bound at subdomain IIA by multiple interactions, such as the interaction between O1 of PFDoA with Arg 257 and Ser 287 predominately through polar force. And the best calculated docking energy is -25.87 kJ·mol-1, this high negative value indicated that the PFDoA molecule exhibited large binding affinity towards HSA. The effects of PFDoA on the conformation of HSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of the microenvironment around tryptophan residue was increased and the conformation of HSA was altered after binding PFDoA. The CD spectra quantitatively calculated the protein secondary structure, which suggested a loss of helical stability after the PFDoA-HSA complex formation. The binding research presented in this paper enriches our knowledge of the interaction dynamics of perfluoroalkyl acids to the HSA and reveals the chemical essence of the interaction between biomacromolecule and ligand.