The interaction between Vitamin B1, thiamine hydrochloride (TAH), and bovine serum albumin (BSA) was studied by spectroscopic and computational methods. The native fluorescence of BSA was quenched by TAH. Stern-Volmer quenching constant was calculated at different temperatures which suggested a static mechanism. The association constant (Ka) was calculated from fluorescence quenching studies, which decreased with temperature rising. TAH competed well with warfarine for hydrophobic subdomain IIA (Sudlow’s site I) on the protein. Enthalpy and entropy changes during the interaction of TAH with BSA were obtained using van’t Hoff plot, which showed an entropy - driven process and involvement of hydrophobic forces (ΔHº > 0 and ΔSº > 0). Optimized docked model of BSA-TAH mixture confirmed the experimental results.