Bio-oil, derived from biomass, offers a sustainable alternative to petroleum-based asphalt binders in construction. However, its high oxygen content and temperature sensitivity pose challenges. This study explored the possibility of using emulsification technology to produce and apply emulsified bio-asphalt at a relatively low-temperature, aiming for sustainable high-value utilization. Three preparation processes were proposed in this study, including modification followed by emulsification (Process A), emulsification followed by modification (Process B), and separate emulsification followed by mixing (Process C). Based on the thermal characteristics of bio-oil, the optimal emulsification temperature was determined to be 80 ± 1 ℃. Through an I-optimal experimental design combined with response surface methodology (RSM), the influence of bio-oil and emulsifier on the performance of emulsified bio-asphalt was investigated for each process. It was found that Process C can leverage the low-temperature extensibility and interfacial adhesion benefits of bio-oil to prepare stable emulsified bio-asphalt with superior comprehensive performance. Based on desirability optimization methodology, the study optimized bio-oil and emulsifier content. The recommended composition is 10.37% bio-oil and 3.53% emulsifier for Process C. Through practical observation, emulsified bio-asphalt production offered environmental benefits, reducing emissions of CO2 and harmful gases, particularly VOCs and NOx. Additionally, adopting bio-oil aligned with carbon neutrality goals, potentially sequestering 880,000 tons of carbon annually in China's road construction and maintenance activities.