Straw return and biochar addition are promising approaches to enhance carbon sequestration and reduce greenhouse gas emission; however, their effect on CO₂-fixing autotrophs remains unclear. Hence, quantitative PCR based on cbbL and cbbM, accC, and hcd, which are marker genes of the Calvin cycle, the 3-hydroxypropionate cycle, and the 4-hydroxybutyrate cycle, respectively, were used to determine the abundance of CO₂-fixing autotrophs in paddy soil. Soils with chemical fertilizers receiving no exogenous carbon amendment, low and high amounts of rice straw, or low and high amounts of biochar were sampled at the tillering, milky, and ripening stages of late rice season. Results showed that both straw and biochar application increased the abundance of CO₂-fixing microbes in paddy soil, whereas more abundant CO₂-fixing microorganisms were observed in HS and LC rather than other treatments. The effects of different amounts of straw and biochar are attributable to the prominent differences in their properties. Redundancy analysis revealed that redox potential, C/N ratio of input fertilizer, and soil content were significantly correlated with the gene abundance of CO₂-fixing microorganisms. Path analysis revealed that gene abundance was causally and indirectly driven by rice growth stage and exogenous C addition. Collectively, our study suggested that adding extra carbon affected the growth of CO₂-fixing microorganisms, which provided novel insights into the effect of straw return and biochar addition in paddy soil.