In this work, the binding mechanism between donepezil (DNP) and bovine serum albumin (BSA) was established using several techniques, including fluorimetry, UV- spectrophotometry, synchronous fluorimetry (SF), fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET) besides molecular docking study. The fluorescence quenching mechanism of DNP-BSA binding was a combined dynamic and static quenching. The thermodynamic parameters, binding forces, binding constant, and the number of binding sites were determined using a different range of temperature settings. Van't Hoff's equation was used to calculate the reaction parameters, including enthalpy change (ΔH^ο) and entropy change (ΔS^ο). The results pointed out that the DNP-BSA binding was endothermic. It was shown that the stability of the drug-protein system was predominantly due to the intermolecular hydrophobic forces. Additionally, the site probing method revealed that subdomain IIA (Site I) is where DNP and BSA's binding occurs. This was validated using a molecular docking study with the most stable DNP configuration. This study might help to understand DNP's pharmacokinetics profile and toxicity as well as provides crucial information for its safe use and avoiding its toxicity.