Perfluorocarboxylic acids (PFCAs) have been widespread used for over half century as surfactants in commercial and industrial products because of their hydrophilic and hydrophobic peculiarity. Perfluoroundecanoic acid (PFUnA) and perfluorotridecanoic acid (PFTriA) are two representatives of long-chain PFCAs, and they were detected more frequently in human body recent years, however, the two PFCAs were found to express endocrine disruption effects, developmental toxicity and teratogenicity. In this study, we established a new strategy to probe the binding modes of PFUnA (PFTriA) with the most abundant protein human serum albumin (HSA) based on spectroscopic and molecular docking methods. Results showed that both PFUnA and PFTriA can quench the intrinsic fluorescence of HSA with one binding site by means of dynamic and static quenching procedure with a strong affinity and the order is PFUnA>PFTriA. On the basis of thermodynamic results, we knew that the main driving force of the interaction between PFUnA and HSA was electrostatic force (ΔH=-26.32 kJ·mol-1, ΔS=21.76 J·mol-1·K-1), while van der Waals interaction and halogen-bond played major roles in complexation process of PFTriA-HSA (ΔH=-39.69 kJ·mol-1, ΔS=-25.66 J·mol-1·K-1). The binding distance (r<8 nm) indicated that the non-radioactive energy transfer came into being from HSA to PFUnA (PFTriA). The binding process of PFUnA (PFTriA) with HSA caused conformational and some micro-environmental changes of HSA, but also led to a loss of helical stability through three-dimensional fluorescence and circular dichroism spectra (CD). Furthermore, site markers competitive experiments and molecular docking revealed that PFUnA and PFTriA had a high affinity into hydrophobic pocket of subdomain IIA in HSA through polar force, hydrophobic interaction and halogen-bond and so on, and the fluorophore Trp residues was located in the binding position which proved further the quenching of PFUnA and PFTriA on HSA fluorescence. The accurate and full basic data in the work are beneficial to clarify the binding mechanism of long-chain perfluorocarboxylic acids with serum protein in vivo, and provide essential theoretical clues for their toxicity assessment and toxicologic research.