This study explores the synthesis and characterization of a bio-based polyol derived from epoxidized cottonseed oil, shedding light on transformative changes in physical and chemical attributes. Executed meticulously in triplicate, the experiment encompasses cottonseed oil epoxidation, Gel Permeation Chromatography (GPC), acid value titration, and Fourier Transform Infrared (FTIR) analysis. Results confirm a successful epoxidation process, yielding an epoxidized oil content (EOC) of approximately 5.88%. Acid values for cottonseed oil (CO), epoxidized cottonseed oil (EO), and polyol (PO) stand at 1.55 mgKOH/g, 1.440 mgKOH/g, and 1.88 mgKOH/g, respectively, indicating notable chemical transformations. FTIR analysis supports the conversion of epoxidized cottonseed oil into polyol, while GPC results affirm precise separation of polyol, epoxidized oil, and cottonseed oil components, signifying successful synthesis. The thorough analysis of polyol properties unveils distinctive changes, including increased viscosity at 25°C from 1.45 Pa.s (cottonseed oil) to 3.901 Pa.s (polyol), denoting enhanced molecular complexity. Additionally, the integration of 2% titanium dioxide (TiO2) filler underscores the potential for modifying and enhancing polyol properties. These findings enrich the understanding of polyol adaptability and versatility, constituting a pivotal advancement in comprehending sustainable and renewable sources for polyurethane production, with profound implications for polymer science and materials engineering.