Interest in the biotoxicology of uranium resulting from its inherent radioactive as well as chemical properties has been growing intensely in recent years. Indeed, uranium in its stable form as UO22+ species is ubiquitously found on earth, and this form is commonly known as the uranyl(VI) ion. The unusual electronic environment at the core of the uranyl(VI) complex plays an important role in its interaction with biomacromolecules. Based on the spectroscopic and computational studies, we have explored the interaction of the uranyl(VI) complex with BSA. The results showed that the fluorescence intensity of BSA was quenched upon interaction with the uranyl(VI) complex largely through dynamic mode, which was authenticated by Stern-Volmer calculations and fluorescence lifetime measurements at different temperatures. Fluorescence anisotropy and synchronous fluorescence spectroscopy were performed to understand the micro-environments of the fluorophores. Furthermore, the binding constant, standard free energy and number of binding sites were also calculated. Thermodynamic parameters such as ΔH° and ΔS° revealed that the non-covalent interactions played a principal role in the binding of the uranyl(VI) complex to BSA, and the value of ΔG° indicated the spontaneity of the interaction. Using the site marker fluorescent probes, the binding location of the uranyl(VI) complex at the BSA site was established. This was further supported by the molecular docking technique with a docking free energy of -38.91 kJ mol-1, indicating the non-covalent binding of the uranyl(VI) complex with BSA active sites. This piece of work may contribute mostly to understanding the pharmacokinetics of the uranyl(VI) complex and provide fundamental data on its safe usage.