The interaction between three isoquinoline alkaloids, berberine, palmatine and coralyne, and the protein lysozyme was studied using fluorescence, absorption, circular dichroism and isothermal titration calorimetry under physiological conditions. The three alkaloids caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism, but with differing quenching efficiencies. The binding constants (K) at 25 °C are 5.37 × 10⁴, 4.22 × 10⁴, and 1.15 × 10⁵ M⁻¹, respectively, for berberine, palmatine and coralyne with binding sites (n) of approximately 1. We have demonstrated strong conformational changes in the secondary structure of the lysozyme molecule on alkaloid binding using synchronous fluorescence spectra, 3D fluorescence results and circular dichroism spectroscopic measurements. Interestingly, binding of the positively charged lysozyme to the positively charged alkaloid was endothermic and entropy driven. The negative standard molar Gibbs energy change (ΔG^o) in all the cases revealed that the binding process was spontaneous. The corresponding ΔH^o and TΔS^o values were 0.58 ± 0.03, 7.09 and 2.37 ± 0.03, 8.67 and 4.31 ± 0.03, 11.23 kcal mol⁻¹, respectively, for berberine, palmatine and coralyne. The thermodynamic parameters (ΔH^o and TΔS^o) of the reaction further indicated that both van der Waals forces and hydrogen bonds play a key role in the interaction. Spectroscopic evidence suggests that Trp62 and Trp63 in the β-domain of the protein are closer to the binding site of the alkaloids. Based on the Forster's theory of non-radiation energy transfer, the binding distances (r) between donor (protein) and acceptor (berberine, palmatine and coralyne) are 3.30, 3.09 and 3.06 nm, respectively. The results provide some valuable information on the interaction of these ligands with the protein.