<p>Polyglycolic acid (PGA)-based materials are frequently exposed to alcohol environments in biomedical and related applications. Although alcohols can significantly influence the macroscopic properties of PGA-based materials, the underlying molecular mechanisms remain elusive. In this study, atomic force microscopy-based single-molecule force spectroscopy (SMFS) was employed to systematically investigate how monohydric alcohol solvents affect the single-chain conformation and mechanical response of PGA, with its inherent elasticity used as a benchmark. SMFS experimental results show that the single-chain elasticity of PGA is highly consistent across different monohydric alcohols but deviates moderately from its inherent elasticity. Density functional theory calculations further demonstrate that both the ester carbonyl oxygen and the ester alkoxy oxygen in each PGA repeating unit can interact with those solvent molecules <i>via</i> hydrogen bonds, which are likely to generate steric crowding around the polymer backbone, thereby promoting chain expansion toward a more extended conformation and ultimately altering the single-chain mechanical response. These findings elucidate the single-molecule mechanism underlying monohydric alcohol-regulated PGA chain conformation and provide guidance for optimizing PGA-based materials in alcohol-containing environments.</p>