The demand for bituminous materials is continuously growing; crude oil-based asphalt binders are non-renewable, and are facing rapid depletion. With the increase of petroleum-based asphalt prices, seeking an alternative, renewable material such as bio-asphalt has become a hot research topic. However, shortcomings in this research area have been identified, notably concerning the high-temperature performance of bio-asphalt at present. This research aims to comprehensively apply conventional tests to, and study the rheological behavior of, the high-temperature performances of bio-asphalt binders, i.e., by temperature and frequency sweeps, using a dynamic shear rheometer (DSR). It will also assess the chemical functional groups of specimens prepared by different aging conditions. Fifty penetration grade base asphalt binder (50#), bio-oil modified asphalt binders with 0%, 5%, 10%, and 30% bio-oil contents by mass, and bio-oil modified asphalt binder with combinations of 5% bio-oil-1% SBS, and 10% bio-oil-1% SBS were used in this study. The conventional performance of bio-asphalt binders was tested using penetration, ductility, and softening point, before and after short-term aging conditioning. The temperature sweep and frequency sweep of bio-asphalt under different bio-oil contents were carried out via DSR. Two-logarithmic equations of rutting factor and temperature were established, and the temperature sensitivity of bio-asphalt was analyzed. The master curves of virgin asphalt and bio-asphalt were constructed at 64 °C. The results indicate that the incorporation of bio-oil reduced the anti-rutting performance of asphalt, and the bio-oil content had a significant effect on the mass loss of the bio-asphalt binder. The performance of bio-oil modified asphalt binders using 5% bio-oil, 5% bio-oil-1% SBS, and 10% bio-oil-1% SBS, could meet the requirements of 50# grade asphalt. The temperature sensitivity of bio-asphalt did not show obvious change before and after short-term aging, whereas the temperature sensitivity of bio-asphalt with 5% bio-oil was relatively small. With an increase in temperature, the phase angle increased gradually. In contrast, the storage modulus, loss modulus, and complex modulus decreased progressively. The complex modulus and rutting factor of bio-asphalt with 5% bio-oil steadily increased with the increase in testing frequency. Otherwise, chemical reactions were detected in the 50# base asphalt modified with the bio-oil.