Space special environment can cause potential damage to astronauts, thus the monitoring of physiological indicators is crucial for the study of damage mechanisms and means of protection. Microgravity as one of the space special environments can lead to mitochondrial dysfunction. Meanwhile, mitochondrial membrane potential is an important indicator of normal mitochondria, it is meaningful to monitor mitochondrial membrane potential under simulated microgravity (SMG) quickly and easily. In this work, a mitochondria-targeting aggregation-induced emission (AIE) probe (TPE-Ph-In) is developed to monitor mitochondrial membrane potential under SMG. At the same time, in order to overcome the problem of insecure cell apposition under a prolonged time of SMG, an AIE probe-hydrogel 3D imaging system is constructed by seeding the cells into Matrigel and imaging the cells with TPE-Ph-In. This work provides a new method and idea to investigate the microgravity effect of cells.