Mass transfer of bubbles is important in microalgae cultivation. In this study, aiming to improve the microalgae culturing efficiency, computational fluid dynamics (CFD) was adopted to study the influence of the aeration manners on mass transfer of bubbles within ascending columns in multi‐column airlift photobioreactor (PBR) and the hydrodynamic conditions within the PBR under different aeration manners. In addition, the bubble generation time, the detachment diameter, and the average volumetric mass transfer coefficient of bubbles in ascending columns were analyzed. Furthermore, the experimental results were compared with the simulation results obtained from microalgae cultivation. The results showed that the whole aeration manner yields the lowest mixing strength and mass transfer efficiency of bubbles at an aeration rate of 0.2 vvm. Conversely, both the mixing strength of the liquid and mass transfer coefficient of bubbles were enhanced under the half and alternate aeration manners. However, the results demonstrated that the distribution of the flow field was not uniform under the half aeration manner and there were obvious high‐speed and low‐speed zones. In contrast, the flow field distribution in the PBR was more uniform under the alternate aeration manner, which was suitable for microalgae cultivation at high concentration. This study effectively enhanced the mixing strength and CO2 transfer rate in the PBR.