Herein we report the binding interactions between lysozyme (Lyz) and an anthracycline drug, epirubicin hydrochloride (EPR), through an extensive spectroscopic approach at both ensemble average and single molecular resolution. Our steady-state and time-resolved fluorescence spectroscopy reveals that the drug-induced fluorescence quenching of the protein proceeds through a static quenching mechanism. Isothermal titration calorimetry (ITC) and steady-state experiments reveal almost similar thermodynamic signatures of the drug-protein interactions. The underlying force that plays pivotal roles in the said interaction is hydrophobic in nature, which is enhanced in the presence of a strong electrolyte (NaCl). Circular dichroism (CD) spectra indicate that there is a marginal increase in the secondary structure of the native protein (α-helical content increases from 26.9 to 31.4% in the presence of 100 μM EPR) upon binding with the drug. Fluorescence correlation spectroscopy (FCS) was used to monitor the changes in structure and conformational dynamics of Lyz upon interaction with EPR. The individual association (Kass = 0.33 × 106 ms-1 M-1) and dissociation (Kdiss = 1.79 ms-1) rate constants and the binding constant (Kb = 1.84 × 105 M-1) values, obtained from fluctuations of fluorescence intensity of the EPR-bound protein, have also been estimated. AutoDock results demonstrate that the drug molecule is encapsulated within the hydrophobic pocket of the protein (in close proximity to both Trp62 and Trp108) and resides ∼20 Å apart from the covalently labelled CPM dye. Förster resonance energy transfer (FRET) studies proved that the distance between the donor (CPM) and the acceptor (EPR) is ∼22 Å, which is very similar to that obtained from molecular docking analysis (∼20 Å). The system also shows temperature-dependent reversible FRET, which may be used as a thermal sensor for the temperature-sensitive biological systems.