Bio-oil is a by-product of waste biomass treatment using high-pressure liquefaction, bio-enzymatic, or high-temperature thermal cracking techniques, and can alleviate the shortage of petroleum asphalt resources. This study investigated the production of bio-oil from selected raw materials, including corn stover, castor oil from castor beans, and epoxy vegetable oil from soybeans for the preparation of bio-asphalt. The properties and mechanisms of modification of the three modified bio-asphalts were explored by assessment of basic physical properties, gel permeation chromatography (GPC), temperature scanning (DSR), and thermogravimetric analysis, as well as Semi-Circular Bending (SCB), low-temperature bending, and fatigue tests. Bio-oil was found to improve the low-temperature cracking resistance of the asphalt mixtures, with the improvement further enhanced by changing the blending amount. The bio-oil was observed to negatively affect the low-temperature fatigue performance of the mixes, and the decrease in the n-value indicated that the bio-oil could make the asphalt mixtures less stress-sensitive under repeated loading, with improved anti-fatigue performance of bio-asphalt mixtures observed when the stress ratio was 0.5. Finally, the low-temperature cracking performance index of the corn stover bio-asphalt mixtures was correlated with the gray entropy. The use of the SCB fracture index was found to be more appropriate when using the semicircular bending test to characterize the low-temperature cracking performance of bio-asphalt mixtures.