Various methods for recovering extracellular polymeric substances (EPS)-based biomaterials from wastewater sludge exist. However, the relationships between extraction methods and properties of biomaterials have not been fully explored. In this study, the thermal properties, including activation energy (AE) and thermal decomposition mechanism, of EPS-based biomaterials extracted by different methods have been determined by thermogravimetric analysis integrated with the deconvolution method. Simultaneously, the chemical properties of these biomaterials, such as the extraction yield, chemical composition, and functional groups, have been monitored to clarify the influences of extraction methods. Notably, proteins and humic-like substances have been found as the major components to determine thermal stability and AE. Moreover, the physicochemical method shows significant effects on enhancing extraction yield and AE, with the NaOH and heat methods proving to be outstanding by delivering the highest AE of 300 kJ/mol and a substantial char formation of 24%. The results have demonstrated the significant impact of extraction methods on the thermal stability of EPS-based biomaterials. Moreover, this finding provides insights into the linkages between the properties of EPS-based biomaterials and extraction methods to guide the selection of appropriate extraction methods tailored to specific applications, including flame-resistant materials.