Bio-asphalt has a high potential to be implemented as a petroleum-based asphalt replacement since it is a renewable and environmentally friendly resource that has identical rheological properties to the conventional binder. Excessive crude oil consumption and fluctuating prices urged researchers to adopt alternatives such as bio-asphalts to replace conventional binders. In this study, the beta-carotene bio-oil (CBO) partially replaced the conventional asphalt binder at 5, 10 and 15. Penetration test, rotational viscosity test, specific gravity test, and mass loss after rolling thin film oven (RTFO) test were conducted to determine the physical and rheological properties of bio-asphalts. FTIR test is used to identify the chemical compositions and functional groups of the CBO bio-asphalt. The binder coatability was assessed by static water immersion and water boiling tests. The addition of CBO has lowered the viscosity and specific gravity while increasing the penetration and the mass loss after RTFO. The FTIR analysis shows that the functional groups of CBO bio-asphalt were mainly phenol, alcohols, ketones, aldehydes, and carboxylic acids. The CBO bio-asphalt enhanced the service characteristic in terms of bonding and coatability behavior. The CBO has a high potential for use as a rejuvenator in the production of asphalt mixtures incorporating reclaimed asphalt pavement (RAP), as it improves the mixture�s workability and adhesion characteristics. © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd 2024.