AbstractLignin, one of the most abundant natural polymers, has been extensively studied as an additive in bituminous binders. Even though the lignin improves the overall resistance against oxidative aging of bitumen, it could lead to high thermal cracking sensitivity. In this study, a bio-oil (i.e., rapeseed oil) is implemented in lignin-modified bitumen to ameliorate characteristics, such as the resistance to fatigue and cracking. The long-term aging of bitumens formulated by different proportions of bio-oil was simulated by the pressure aging vessel (PAV) method. Fourier Transform Infrared Spectroscopic (FTIR) results demonstrated lignin has a remarkable antiaging effect, but adding bio-oil will slightly deteriorate the antiaging effect. The Brookfield rotational viscometer showed that the addition of bio-oil was able to reduce the binder’s viscosity observably, thereby improving the workability of bitumen. The frequency sweep tests revealed that lignin increased the stiffness and improved the thermal stability. Also, the multiple stress creep recovery tests corroborated that lignin significantly enhanced the rutting resistance of binders. The fatigue and thermal cracking properties of lignin-modified binder could be strengthened by increasing bio-oil content which is confirmed from both linear amplitude sweep, Glover-Rowe parameter, and Bending Beam Rheometer (BBR) tests. Moreover, the relaxation test results demonstrated that bio-oil decreased the residual stress ratio and relaxation time dramatically. Overall, this study has shown preliminary conclusions on the incorporating of bio-oil to enhance the medium-low temperature properties of lignin-modified binders. In the meantime, bio-oil did not interfere with the improvement influence of lignin on the neat bitumen regarding high-temperature performance.