Biological carbon fixation is crucial to the Earth's carbon cycle and is one of the effective ways to transform CO2 and manage carbon emissions. Chemoautotrophs, with their unique metabolic strategies and environmental adaptability, play an important role in this process. They are able to convert CO2 into valuable organic products, solving the problem of limited CO2 utilization. However, the carbon fixation potential of chemoautotrophs in controlled systems has not been fully explored. This review illustrates the possible challenges of stable culture of chemoautotrophic bacteria in bioreactor. Based on this, a series of physical, chemical and biological methods are proposed to regulate the carbon metabolism of chemoautotrophic bacteria and improve their carbon fixation efficiency. Further, the application prospects of chemoautotrophic carbon fixation in controlled systems are expected, including improving the primary productivity of natural ecosystems, reducing carbon emissions in specific sites, and producing high-value microbial by-products. This review highlights the advantages and challenges of these applications, providing important insights into carbon capture, fixation and conversion by chemoautotrophs.