Recent efforts are being conducted to develop alternative asphalt binders from various bio-mass resources for future flexible pavements construction due to their renewability and the increasing costs of conventional petroleum-based asphalt. The objective of this paper is to investigate the potential of using the waste cooking oil (WCO) based bio-oil as a modifier for petroleum based neat asphalt binder and Styrene-Butadiene-Styrene (SBS) modified binder by means of chemical and rheological approaches. A series of tests were conducted for such purpose, including the infrared spectroscopy test, frequency sweep rheological test, multiple stress creep recovery test, and linear amplitude sweep test. The infrared spectroscopy results indicate identical chemical functional groups between the bio-oil and the petroleum asphalt binder though acid, ether, ester and alcohol compounds were also observed within the bio-oil. The bio-oil modified binders display increased carbonyl index with increasing the bio-oil percent weight whereas the sulfoxide index almost exhibits the same level as that of the control asphalt. Frequency sweep tests show that the bio-oil addition obviously decreased the binder stiffness according to the dynamic shear modulus master curve. Due to this softening effect from the bio-oil modifier, the weakened rutting resistance of bio-binders are demonstrated for both neat and SBS binders at the high temperature range. The fatigue life of bio-binders at intermediate temperature under cyclic fatigue loading are found to be significantly improved by increasing bio-oil content but the binder yield energy simultaneously decreased. It can be preliminarily concluded that the WCO based bio-oil tested in this study could be used as a potential bio-modifier to produce a sustainable asphalt binder.