With the rapid industrial development, massive fossil fuel use has caused excessive carbon dioxide (CO2) emissions, triggering global warming and environmental issues. Thus, CO2 recovery and reuse have become a research focus, among which artificially designed in vitro biocatalytic pathways for converting CO2 into high-value chemicals show promise, with advantages like shorter routes, higher efficiency and lower energy consumption compared to natural pathways. However, challenges remain due to natural enzymes' issues in specificity, affinity, efficiency, stability and oxygen sensitivity. To tackle these problems, extensive research efforts have been undertaken. These include elucidating the mechanisms and catalytic efficiencies of carbon-fixing enzymes from diverse sources, as well as developing and refining novel in vitro carbon fixation pathways. Moreover, significant progress has been made in computer-aided investigations of enzyme structure, function, and engineering optimization, alongside advancements in enzyme immobilization strategies, cofactor regeneration systems, and the development of artificial cofactors. By summarizing the latest research progress in recent years, we can identify the current bottlenecks and challenges in in vitro enzymatic CO2 conversion, propose effective methods to enhance the efficiency of CO2 conversion, and thus promote the development of research in related fields.