Aflatoxin B1, a mycotoxin produced by large number of Aspergillus species including Aspergillus flavus and Aspergillus parasiticus, has been described as the most potent carcinogenic mycotoxin. In this study, we have used a multiple spectroscopic and molecular docking approach to investigate the interaction of aflatoxin B1 (AFB1) with chicken egg albumin (CEA). Fluorescence spectroscopy, UV-Vis spectroscopy, and three-dimensional fluorescence spectroscopic techniques were employed to gain insight into the conformational changes in CEA in the presence of AFB1. Fluorescence spectroscopy revealed ligand-induced quenching in the fluorescence emission spectra of CEA upon binding with AFB1. Hyperchromic effect was observed in case of the ground state complex formation between CEA and AFB1 by UV-Vis spectroscopy. To gain further comprehension into the site of binding of AFB1 to CEA, competitive site marker displacement assay was performed using warfarin site marker. The magnitude of ΔG value calculated from fluorescence-based method was negative which confirmed spontaneous process. The results obtained suggest that the binding is enthalpy driven and van der Waals force and hydrogen bonds are stabilizing the AFB1-CEA complex. Three-dimensional fluorescence studies also confirmed the quenching in the fluorescence intensity around tryptophan residues in CEA. Circular dichroism assessment revealed reduction in the alpha helical content of CEA in the presence of AFB1. Molecular docking studies showed hydrophobic interaction, van der Waals forces, and hydrogen bonds as major forces present in interaction between CEA and AFB1. The overall study confirms conformational and structural alteration in the protein due to binding of AFB1.Communicated by Ramaswamy H. Sarma