The microalgae-based CO₂ sequestration is considered to be an effective technique with great potential to cope with carbon emission. However, most researches are only focused on microalgae; the effects of physicochemical factors, which are carbon concentration, medium pH, and bubbling depth, on absorption and utilization of supplied CO₂ in culture is less known. In order to understand and improve CO₂ absorption in microalgae culture, the effects of these three factors were studied with different levels and combinations. Results revealed that when medium carbon concentration increased from 4.76 to 95.24 mmol/L, CO₂ absorption ratio increased by about 12%, 10%, 12%, and 11% at medium depths of 10, 20, 40, and 80 cm, with the initial pH 10.6 to 9.7 by bubbling CO₂, respectively. As bubbling depth increased from 10 to 80 cm, CO₂ absorption ratio increased by about 25%, 22%, and 25% at carbon concentrations of 4.76, 9.52, and 95.24 mmol/L, with the initial pH 10.6 to 9.7 by bubbling CO₂, respectively. In range of 10.6-7.0, pH had no significant effect on CO₂ absorption ratio (P > 0.05) when carbon concentration is below 9.52 mmol/L, while above 9.52 mmol/L, pH had significant effect on CO₂ absorption ratio (P < 0.05). It was found for the first time that the effect of pH on the CO₂ absorption ratio was affected by carbon concentration. In addition, equilibrium pH, at which the CO₂ partial pressure in the medium equals to that in the air, of medium with different carbon concentrations was also determined. Overall, in microalgae culture for CO₂ sequestration, increasing CO₂ bubbling depth and keeping higher carbon concentration and higher pH can improve CO₂ absorption ratio, which will optimize the biofixation of CO₂ by microalgae furthermore.