Danofloxacin (DNF), a synthetic fluoroquinolone, is widely used as an antibacterial agent against a broad spectrum of pathogens. In the present study, the effects of DNF on the structure of bovine serum albumin (BSA) were investigated using UV-Vis absorption and fluorescence spectroscopy, and molecular docking methods at different temperatures. The obtained results of UV-Vis absorption studies showed that the microenvironment of the fluorophore residues does not significantly change upon interaction with DNF. Also, fluorometric studies revealed BSA-DNF complex formation and fluorescence quenching of BSA in the presence of DNF. The number of binding sites and binding constants were calculated to be ~1 and in the order of 103, respectively. According to thermodynamic parameters, van der Waals forces and hydrogen bonding play the main role in the BSA-DNF complex formation, which is a spontaneous process. The binding distance between DNF and BSA was calculated by the Förster resonance energy transfer (FRET) method. Molecular docking results were in agreement with thermodynamic and spectroscopic data and confirmed the binding mechanism of DNF to BSA.