The chemical and rheological properties of polymer modified bitumen incorporating bio-oil derived from waste cooking oil (WCO) were investigated in this paper. At first, the chemical composition and mixing mechanism of the experimental materials were analysed from the perspective of functional group, and the influence of bio-oil on the activation energy was also researched. Then, the effect of bio-oil on the rotational viscosities of polymer modified bitumen and construction temperatures of corresponding mixtures was studied. Finally, the shear and bending rheological properties of polymer modified bitumen containing bio-oil were investigated. The results show that the bio-oil and styrene–butadiene–styrene (SBS) modified bitumen is mainly physically mixed, the addition of bio-oil decreases the activation energy of SBS modified bitumen. Additionally, the SBS modified bitumen containing bio-oil has lower viscosity values, and corresponding mixtures also have lower construction temperatures. Furthermore, the addition of bio-oil in SBS modified bitumen reduces the shear modulus and increases the bending creep compliance, which means bio-oil has positive effect on the low-temperature thermal cracking resistance performance while sacrificing the high-temperature rutting resistance performance to some extent. Therefore, the incorporation of WCO-based bio-oil in polymer modified bitumen is a promising technique to be used in cold regions where the low-temperature problems are more crucial.@en