With the deepening of exploration in Santos Basin, Brazil, it is found that CO2 is the main geological risk of pre-salt oil and gas accumulation. At present, there is a lack of systematic research on the formation mechanism and the distribution law of CO2 in the basin. In this paper, the formation controlling factor of CO2 was explored by geochemical analysis of the samples from wells and characterization of the fault system based on geophysical data. The CO2 content with a wide range of variation in the Santos Basin accounts for 0-96， of the gas content in the discovered oil and gas reservoirs. It shows the CO2 content is low in the western area of basin (commonly less than 1，), and high in the eastern area of basin (generally more than 30，). CO2 gas caps and CO2 gas reservoirs had been discovered in the eastern part of basin. The δ13C of CO2 in the basin is -8.64‰- -7.03‰, and the helium isotope ratio R/Ra is about 5.6-8. The CO2 in the Santos Basin originated from the mantle, and was basically not polluted by the crust. The geological background of CO2 accumulation was the uplift of the mantle. The thinning of the crust caused easily volcanoes eruption on the surface in the eastern part of the basin, causing the release of deep CO2. Main controlling factors of CO2 accumulation were deep faults. The dominant migration channels were the intersection of extension and strike-slip faults (four groups of fractures), especially the reactivated faults during the drift period, which directly or indirectly communicating with mantle-derived magma chambers, and a large amount of mantle source CO2 migrated to the sedimentary strata to form oil and gas reservoirs with high CO2 content or CO2 gas reservoirs. This research result guided the successful acquisition and exploration breakthrough of the A block.