This paper investigates the use of a plant-based bio-oil in bituminous binders as a partial replacement of petroleum-based bitumen for asphalt mixtures. Its effects on the ageing behaviour of bituminous binders and asphalt mixtures are studied. A total of six bituminous binders and their asphalt mixtures were prepared and analysed in laboratory, including three different binder formulations with varying percentages of bio-oil and their respective reference binders. Both the bituminous binders and asphalt mixtures were subjected to ageing protocols in laboratory. Softening point test, rheological and dynamic mechanical analyses were conducted to evaluate the changes in properties of the materials before and after ageing. The results indicate that the mechanical properties of aged binders and mixtures show very similar relationships as between the fresh materials, but the relationships after ageing are at changed levels due to the laboratory conditioning. This supports further studies to verify their functional performance in asphalt pavements.