In recent years, in order to deal with the excessive CO2 emission caused by fossil fuel combustion and the resulting global warming and energy depletion, CO2 mitigation strategy via carbon fixation and conversion to high-value products by microalgae cultivation system has attracted a great deal of attentions. In particular, the addition of CO2 absorbents has been reported effective to promote microalgal growth and carbon fixation rates. In this study, Chlorella sp. with excellent CO2 tolerance was used as the experimental strain to explore the effects of chemical adsorbent monoethanolamine (MEA) on algal physiological and biochemical characteristics and carbon sequestration efficiency under 20% CO2 concentration with ventilation rate of 0.33. The results showed that adding MEA could alleviate the growth inhibition of microalgae by culture medium acidification caused by high concentration CO2. In addition, MEA with appropriate concentration could also effectively improve the growth, metabolism, CO2 fixation efficiency and photosynthetic activity of Chlorella sp. The biomass concentration, CO2 fixation rate and lipid content of Chlorella sp. all reached maximum value of 3.07 g/L, 0.55 g CO2/(L·d) and 23.5% respectively under 50 mg/L MEA, which were 43.7%, 45.6% and 21.7% respectively higher than those without MEA in control. In summary, 50 mg/L CO2 absorbent monoethanolamine could significantly improve the biomass, lipid accumulation and carbon sequestration efficiency of Chlorella sp. under high CO2 concentration. The above results would provide theoretical basis for CO2 emission reduction and clean energy development by microalgae.