•A fatigue simulation algorithm based on WRsum failure criterion was validated for bio-binders.•Bio-oil modification reduced binder stiffness but improved resistance to fatigue cracking.•Aging enhanced binder deformation resistance but compromised fatigue performance.•Consistent observations on fatigue were made from rheological parameters and LAS approach. This study was aimed to evaluate the fatigue characteristics of asphalt binders modified with different dosages of bio-oil under a variety of aging conditions. The frequency sweep test was performed to determine four rheological parameters indicative of cracking resistance. The linear amplitude sweep (LAS) test was conducted to yield damage characteristic relationship, failure criterion, and failure envelope based on the viscoelastic continuum damage theory. In general, consistent observations were obtained between the rheological parameters and the LAS-based fatigue analysis, in that higher percentages of bio-oil in modification provided improved resistance to cracking, while higher aging intensities produced lower fatigue performance.