Recent studies have increasingly focused on molecular interactions between small molecules and proteins, especially binding mechanisms and thermodynamics, using multispectroscopic and molecular dynamics approaches. This study elucidated the molecular interaction mechanism between bovine serum albumin (BSA) and trans-resveratrol (Res) through an integrated approach combining multispectroscopic analyses and molecular dynamics simulations. The fluorescence quenching study revealed a static quenching mechanism between BSA and Res, which was further confirmed via ultraviolet–visible (UV-Vis) absorption spectroscopy. In particular, K_SV decreased from 5.01 × 10⁴ M⁻¹ at 298 K to 3.99 × 10⁴ M⁻¹ at 318 K. Furthermore, the calculated K_q values significantly exceeded 1 × 10¹² M⁻¹ s⁻¹. With increasing Res concentration, the peak fluorescence intensities of Tyr and Trp residues both exhibited a blue shift. The α-helix content of the BSA–Res complex was 59.8%, slightly lower than that of BSA (61.3%). Res was found to bind to site I in subdomain IIA of BSA. The molecular dynamics simulation also identified the specific binding of Res to site I of BSA, while thermodynamic studies revealed that the binding process occurs spontaneously and is primarily mediated by hydrogen bonding interactions. These findings not only enrich the theoretical framework of small-molecule–protein interactions but also provide a crucial scientific foundation for the development and utilization of natural products.