Microbial CO₂ fixation is a promising pathway of carbon capture and utilization, which is crucial for achieving carbon neutrality in global ecosystems. Non-photosynthetic autotrophic microorganisms have attracted attention for their environmental adaptability, diversity of carbon fixation pathways, and production of high value-added products. Based on domestic and foreign researchs on CO₂ fixation by non-photosynthetic autotrophic microorganisms, we systematically analyzed internal and external factors affecting the carbon fixation efficiency of non-photosynthetic autotrophic microorganisms, including CO₂ fixation pathways, key carbon fixing enzymes, carbon sources, electron donors, and metabolites. Various strategies for promoting microbial CO₂ fixation have been proposed. In addition to strategies involving direct responses to influencing factors, strategies involving synergy between microorganisms and the use of metabolic promoters (biochar and micro/nanobubbles) are also being investigated. This review provides a theoretical foundation and technical support for enhancing CO₂ fixation by non-photosynthetic autotrophic microorganisms.