Bio-asphalt binders have been proposed as replacements for traditional asphalt binders, owing to advantages such as environmental protection, low costs, and abundant resources. However, a limitation of bio-asphalt binders is that their high-temperature performance is not suitable for pavement construction. In recent years, nano-particles have been widely used to improve the pavement performance of asphalt binders, particularly the high-temperature performance. Thus, the nano-particles might also provide a positive modified effect on the high-temperature performance of bio-asphalt binders. Based on this, five types of nano-particles including SiO2, CaCO3, TiO2, Fe2O3, and ZnO are selected for the preparation of modified bio-asphalt binders, using different dosages of nano-particles and bio-oil. The high- and low-temperature performances, aging resistance, workable performance, and water stability of the nano-modified bio-asphalt binders and mixtures are investigated. The results reveal that, the high-temperature performance and aging resistance of the nano-modified bio-asphalt binders and mixtures are improved at increased nano-particle dosages, whereas their low-temperature performance is slightly weakened. The effects of the nano-particles on the workable performance and water stability are insignificant.