Ciprofloxacin (CPFX) and enrofloxacin (ENFX) are two of the most widely used fluoroquinolones (FQs) in human and veterinary medicines. Their occurrence in the environment has received much attention because of the potential adverse effects on humans and ecosystem functions. In this paper, we investigated the interaction mechanism between the two FQs and lysozyme by the spectroscopic and molecular docking methods. As shown by the fluorescence spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of lysozyme, which was attributed to the formation of a moderately strong complex. The enthalpy change (ΔH) and entropy change (ΔS) indicated that van der Waals forces and hydrogen bonds were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore, data obtained by UV-vis absorption, synchronous fluorescence and circular dichroism (CD) suggested that both CPFX and ENFX could lead to the conformational and some microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two FQs had specific interactions with the residues of Trp62 and Trp63.