This research investigates the efficacy of bio-oil as a sustainable modifier for lignin-modified asphalt (LMA), aiming to enhance its performance characteristics. Utilizing Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), the study analyzes the chemical and microstructural changes induced by bio-oil in LMA. Rheological properties were evaluated using Dynamic Shear Rheometry (DSR), revealing that the addition of 5-10% bio-oil to LMA significantly reduced stiffness and brittleness, improving ductility and fatigue resistance. For instance, LMAs with 10% bio-oil demonstrated a fatigue life at 2.5% strain comparable to unmodified asphalt. Additionally, bio-oil inclusion increased adhesive strength between asphalt and aggregates, enhancing moisture resistance. Low-temperature properties assessed by dynamic mechanical analysis (DMA) showed improved flexibility and thermal crack resistance with bio-oil addition. These findings underscore the potential of bio-oil in developing high-performance, sustainable asphalt binders, contributing to the advancement of eco-friendly road construction materials.