In order to promote photosynthetic reactions and biomass yield of microalgae in 15% CO2 from coal-fired flue gas, a self-adaptive microalgal growth system with gene mutation centered on the cellular light response was established through CO2 gradient domestication. The microalgae Nannochloropsis oceanica CCMP1779 was gradually acclimated to 0.04%, 3%, 6%, 10% and 12% CO2 to obtain strains with a predominant adaptation to CO2. The domesticated strain showed a 22.7-fold and 3.98-fold increase compared to wild-type under 6 mL/min and 12 mL/min 15% CO2, respectively. A comprehensive analysis of the genome, transcriptome, photosynthetic fluorescence and RT-qPCR results were conducted to explore the effects of CO2 gradient acclimation on microalgal growth. The CO2 gradient domestication produced multi-targeted genetic mutations and natural selection, leading to successful transcriptional changes and apparent photosynthesis properties. Therefore, a self-adaptive microalgal growth system for high CO2 was built based on photosynthesis enhancement (controlled by magnesium chelatase (EC 6.6.1.1)) in the competition between photosynthesis and respiration. This system was supported by improved cation transport, DNA and RNA synthesis, photosynthetic electron transfer, carbon fixation and metabolism. CO2 gradient acclimation demonstrates a cost-effective method for generating microalgae mutants for efficient CO2 fixation from coal-fired flue gas.