<p>Developing sustainable partial replacement strategies for petroleum-based binders is essential to enhance the durability, performance, and environmental sustainability of flexible pavements. In this study, treated rubber seed oil was used as a bio-binder in combination with crumb rubber (15% by weight of base binder) to develop a composite bio-asphalt. The conventional asphalt binder was partially replaced with bio-binder at three dosage levels of 3%, 5%, and 7%. The chemical characteristics of the bio-oil, bio-binder, and crumb rubber were first characterized, followed by a comprehensive evaluation of the physical, chemical, and rheological properties of the composite binders. Subsequently, the optimum binder content (OBC) for each asphalt mixture was determined using the Marshall mix design method. Asphalt mixtures prepared at their respective OBCs were evaluated for mechanical performance and durability using indirect tensile strength, tensile strength ratio, and static creep tests to assess the strength characteristics, moisture damage resistance, and permanent deformation behavior, respectively. The experimental results demonstrated that the rutting performance of the composite bio-asphalt containing 15% crumb rubber and 5% bio-binder was comparable to that of conventional asphalt at both the binder and mixture levels, indicating its strong potential as a sustainable alternative binder for flexible pavement applications.</p>