In this paper, the fluorogenic property of vindoline was exploited and, as a probe, used to analyze the interaction of vindoline with HSA by fluorescence and absorption spectra in combination with molecular modeling under a simulated physiological conditions.  The evidences from synchronous fluorescence and    absorption spectroscopes showed the effect of vindoline on the microenvironment around HSA in aqueous    solution.  Data obtained by the fluorescence spectroscopy indicated that binding of vindoline with HSA leads to dramatic enhancement of the fluorescence emission intensity.  The binding constants and the number of binding sites between vindoline and HSA at different temperatures (303, 310 and 317 K) were calculated according to the data obtained from fluorescence titration.  Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that vindoline can bind strongly to HSA.  It is considered that vindoline binds to HSA mainly by a hydrophobic interaction and there are four hydrogen bonds interactions between the drug and the residues Ala291, Arg222, Arg218 and Lys195, separately.  Fluorescent displacement measurements confirmed that vindoline bind HSA on site II.  The thermodynamic parameters obtained (the enthalpy change ΔH ⁰ and the entropy change ΔS ⁰ were calculated to be −10.30 kJ·mol ⁻¹ and 79.98 J·mol⁻¹·K⁻¹, respectively,   according to the Van’t Hoff equation) suggested that hydrophobic and electrostatic interaction is the predominant intermolecular forces stabilizing the complex.