CO₂ utilization becomes a promising solution for reducing anthropogenic greenhouse gas (GHG) emissions. Biomass-based CO₂ utilization (BCU) even has the potential to generate negative emissions, but the corresponding quantitative evaluation is limited. Herein, the biomass-based CO₂ utilization with an iron cycle (BCU-Fe) system, which converts CO₂ into formate by Fe under hydrothermal conditions and recovers Fe with biomass-derived glycerin, was investigated. The GHG reduction potential under various process designs was quantified by a multidisciplinary method, including experiments, simulations, and an ex-ante life-cycle assessment. The results reveal that the BCU-Fe system could bring considerable GHG emission reduction. Significantly, the lowest value is −34.03 kg CO₂-eq/kg absorbed CO₂ (−2.44 kg CO₂-eq/kg circulated Fe) with the optimal yield of formate (66%) and Fe (80%). The proposed ex-ante evaluation approach not only reveals the benefits of mitigating climate change by applying the BCU-Fe system, but also serves as a generic tool to guide the industrialization of emerging carbon-neutral technologies.