The study of the structural changes and thermal stabilities of proteins in the presence of various co-solutes is a critical element of basic research, drug discovery, and development. Ethylene glycol (EG) is typically known to be poisonous and teratogenic, causing skeletal and external abnormalities as well as other disorders. At pH 8, the influence of EG on the structure and thermal stability of trypsin was investigated using a variety of spectroscopic techniques. Spectroscopic techniques, fluorescence spectroscopy, circular dichroism (CD), and molecular dynamics simulation studies were used to evaluate the binding reaction between ethylene glycol and trypsin in the current study. Circular dichroism, Trp fluorescence, and absorption tests demonstrated that EG has an effect on secondary and tertiary structures of trypsin, and it boosts the protein's thermal stability, which increases with increasing EG concentration. This contact occurs through a static quenching process, according to fluorescence spectroscopy data, and van der Waals contributions and hydrogen bonds were also important in binding ethylene glycol to trypsin. The ethylene glycol binding constant (Kb) and the number of binding sites (n) were calculated using fluorescence quenching data. Kinetic technique analysis displayed a decrease in trypsin activity in the presence of ethylene glycol. Also, circular dichroism results confirmed the conformation changes resulting from the binding of ethylene glycol to trypsin. Molecular dynamics simulation using RMSD was used to test the stability of the trypsin-ethylene glycol complex in this study. Molecular docking research revealed that EG interacts with trypsin amino acid residues via hydrogen bonding and van der Waals interactions.