The binding mechanism between ethyl viologen (EV) herbicide and lysozyme (Lys) was studied using spectroscopies and molecular docking. Apparent association constant (5.04 × 104 L/mol) was calculated using UV–Vis study and suggested the formation of a complex between Lys and EV. Fluorescence quenching of Lys occurred via a static quenching as confirmed by time-resolved data. Binding constant obtained using temperature dependent fluorescence quenching and strong binding affinity (15.8 ± 0.12 × 104 L/mol at 298 K) between Lys and EV has been observed. The mode of interaction studied using thermodynamic parameter, and weak force is responsible for the formation of Lys-EV complex. The binding distance between EV and Lys was found to be 1.57 nm indicating a non-radiative energy transfer process. There is no clear evidence of significant changes in the structure of Lys in the presence of EV. Also, experimental results for the Lys-EV interaction were in agreement with those finding of theoretical simulations.