Bio-oil produced during the production of biodiesel is a burden to the environment. Recycling and utilization of bio-oil as a substitute for pavement bitumen can help to build an environmentally-friendly and clean infrastructure. In this study, the bio-bitumen was prepared by bio-oil based on free radical polymerization. Different kinds of bio-bitumen products were produced by reacting bio-oil with an initiator and an accelerator solution at different reaction conditions. The orthogonal experimental method was employed to determine the optimal bio-bitumen production process by evaluating the indices of viscosity, rutting factors and fatigue factors. The test results show that the optimal mass proportions of bio-oil:initiator:accelerator solution is 100:1:2. Materials with these mass proportions should react at 100 °C for 2 h to yield the best bio-bitumen product. This kind of bio-bitumen product can be considered as a promising substitute for traditional petroleum bitumen.