Applying bio-oil to asphalt pavement effectively reduces the dependence on traditional petroleum asphalt in pavement industry. However, the utilization of bio-oil can encounter rutting and aging issue of modified asphalt. To solve the aforementioned issue, two kinds of organic layered silicates with typical structures were adopted to enhance the high-temperature and anti-aging performance of the bio-modified asphalt. The modification mechanism and the synergistic effect of bio-oil and organic layered silicate on asphalt were explored through rheological characterization, infrared spectroscopy tests, gel chromatography tests, etc. Results showed that the incorporation of layered silicate in bio-modified asphalt can improve asphalt-filler interaction and hence the high-temperature and fatigue performance of asphalt. When the content of layered silicate is 5 %, the negative effect of Castor oil on the high-temperature performance of asphalt can be eliminated. Still, for Turpentine wood oil and Straw oil, the content of layered silicate is 7 %, which shows the same effect. The waste bio-oils can balance the low-temperature performance of asphalt modified with layered silicates. Furthermore, the addition of layered silicates was effective to reduce oxidation reaction and improve the anti-aging performance of bio-oil modified asphalt. When the ratio of layered silicate to bio-oil is 1 1, the composite-modified asphalt maintains the best anti-aging performance. Specifically, Castor oil, Straw oil, and OMMT are more suitable, and Turpentine wood oil and OREC are more appropriate. According to the quantification of the functional group aging index, the SI and CI of the optimal ratio of different types of bio-oil asphalt are Castor oil: 61.64 % and 59.41 %, Turpentine wood oil: 50.02 % and 53.57 %, Straw oil: 50.01 % and 55.05 %. Composite modified asphalt can be applied to complex service conditions.