<p>Asphalt binder replacement with bio-materials (bio-asphalt) has recently gained significant attention. Bio-asphalt serves as a sustainable avenue and is currently being researched for reducing the dependency on asphalt binder. This work is aimed towards understanding the thermal, morphological, chemical, and rheological behaviour of asphalt binder partially replaced with sugarcane molasses (SM). The optimum dosage for partial replacement was arrived as 30% (by weight of asphalt binder). Two base binders (VG 40 and VG 30) along with five SM sources were used to prepare the bio-asphalts. The thermal stability evaluated using thermogravimetric analysis revealed that bio-asphalts have acceptable thermal resistance withstanding temperature up to 200&#xa0;°C. Fluorescence microscopy exhibited that SM particles were uniformly dispersed in the base binder, rendering a stable structure. Through chemical analysis (asphaltene–maltene ratio) it was found that the asphaltene percentage marginally increases after the addition of SM. Rheological characterization comprised of multiple stress creep recovery and linear amplitude sweep tests. Test results indicated that incorporation of SM resulted in lower non-recoverable creep compliance (<i>J</i>_nr), decreased permanent strain, and similar/slightly higher percent recovery (% R). The fatigue life of bio-asphalts improved due to the formulation of compounds capable of imparting elasticity to the bio-asphalts.</p>