Biological CO2 fixation is so far the most effective means for CO2 reduction at scale and accounts for most of the CO2 fixed on Earth. Through this process, carbon is fixed in cellular components and biomass during organismal growth. To uncouple CO2 fixation from growth and cellular regulation, cell-free CO2 fixation systems represent an alternative approach since the rate can be independently manipulated. Here we designed an oxygen-insensitive, self-replenishing CO2 fixation system with opto-sensing. The system comprises a synthetic reductive glyoxylate and pyruvate synthesis (rGPS) cycle and the malyl-CoA-glycerate (MCG) pathway to produce acetyl-coenzyme A (CoA), pyruvate and malate from CO2, which are also intermediates in the cycle. We solved various problems associated with the in vitro system, and implemented opto-sensing modules to control the regeneration of cofactors. We accomplished sustained operation for 6 hours with a CO2-fixing rate comparable to or greater than typical CO2 fixation rates of photosynthetic or lithoautotrophic organisms.