Levofloxacin (LVFX) is widely used in clinical treatment due to it has a broad spectrum of in vitro activity against Gram-positive and Gram-negative bacteria. Human serum albumin (HSA) is the most abundant protein in plasma and constitutes approximately half of the protein founds in human blood. And more than 90% of the drugs used in people are bound to HSA. So it is commonly used for the investigation of drug-serum albumin interaction because the binding will significantly influence the absorption, distribution, metabolism excretion, stability and toxicity of the drugs. Therefore, detailed investigating the interaction of LVFX with HSA is very important to understand the pharmacokinetic behavior of the LVFX. In this paper, the interaction of LVFX and HSA has been studied fluorescence, UV, Fourier transform infrared (FT-IR) and molecular modeling method. The results indicated that LVFX induced the intrinsic fluorescence quenching of HSA though a static quenching procedure, and the effective binding constants (Ka) were calculated to be 9.44×104 L·mol-1 (294 K) and 2.74×104 L·mol-1 (310 K) by used of the Stern-Volmer equation. According to the Vant’s Hoff equation, the reaction was characterized by negative enthalpy (ΔH=-59.00 kJ·mol-1) and negative entropy (ΔS=-105.38 J·mol-1·K-1), indicated that the predominant forces in the LVFX-HSA complex were hydrogen bonding and van der Waals forces. By displacement measurements, the specific binding of LVFX in the vicinity of Site Ⅰ of HSA was clarified. The binding distance of 3.66 nm between Trp214 and HSA was obtained by the Frster theory on resonance energy transfer. Furthermore, the binding details between LVFX and HSA were further confirmed by molecular docking studies, which were consistent with the experimental results. The alternations of protein secondary structure were calculated from FT-IR spectra. Upon formation of LVFX-HSA complexes, the amount of α-helical structures were decrease, but the numbers of β-sheet structures, β-turn structures and random structures were increase, respectively. This result indicated that LVFX induced unfolding of the polypeptides of HSA.