To improve the performance of petroleum asphalt, bio-char was used as a modifier for a petroleum asphalt binder, in this study. The rheological properties of bio-char modified asphalt binders were compared with different particle sizes and contents, with one control and one flake graphite modified asphalt binder. Specifically, the bio-char modifiers with two particle sizes (ranging from 75 μm–150 μm and less than 75 μm) and three contents of 2%, 4%, and 8% were added into the asphalt binder. A flake graphite powder with particle sizes less than 75 μm was used as a comparison modifier. The Scanning Electron Microscopy (SEM) image showed the porous structure and rough surface of bio-char as well as dense structure and smooth surface of flake graphite. A Rotational Viscosity (RV) test, Dynamic Shear Rheometer (DSR) test, aging test, and Bending Beam Rheometer (BBR) test were performed to evaluate the properties of bio-char modified asphalt in this study. Both modifiers could improve the rotational viscosities of the asphalt binders. The porous structure and rough surface of bio-char lead to larger adhesion interaction in asphalt binder than the smooth flake graphite. As a result, the bio-char modified asphalts had better high-temperature rutting resistance and anti-aging properties than the graphite modified asphalt, especially for the binders with the smaller-sized and higher content of bio-char particles. Furthermore, the asphalt binder modified by the bio-char with sizes less than 75 μm and about 4% content could also achieve a better low-temperature crack resistance, in comparison to other modified asphalt binders. Thus, this type of bio-char particles is recommended as a favorable modifier for asphalt binder.