In this study, an advanced noninvasive gamma-ray computed tomography (CT) technique was used for the first time to visualize and quantify the cross-sectional gas holdup distribution of the multiphase flow of an air—water-microalgae system in a cylindrical split airlift photobioreactor. The gas holdup distribution and their radial profiles were investigated during the culturing of the green microalgae Scenedesmus. The gas phase distribution in the entire reactor, including riser region, downcomer region, distribution regions, as well as their distributions above and below the split plate was also characterized, along with the impact of two superficial gas velocities (0.01 and 0.03 m/s), different axial levels, and the changes in the culture from first day until the medium became very dense. The results showed an excellent gas phase distribution in most of the reactor's regions and had high values around the sparger ring and above the split plate. Moreover, the results indicated that the flow distribution may significantly affect the performance of the photobioreactor, which may have substantial effects on the cultivation process. The results are reliable as benchmark data to validate computational fluid dynamics (CFD) simulations and other models.