Tall oil pitch (TOP) is widely available as a by-product, but not sufficiently valorized and has potential as a bitumen extender. In this research, three types of TOP were used to prepare bio-extended binders of two grades based on the penetration and softening point of target neat binders. The chemical, rheological, and fatigue behaviour of bio-based binders and reference binders were investigated and compared. The optimum TOP contents were inversely determined based on penetration and softening point fitting equations. New C = O and C–O–C stretching can be found in bio-based binders. Different TOPs can significantly increase the viscous response of asphalt binders, resulting in better low-temperature properties but reduced high-temperature properties. A high amount of TOP can significantly reduce fatigue resistance. This work reveals that TOP can work as extenders for bitumen, but the performance of bio-based binders is affected by the TOP type and amount.