Space environment can cause damage to astronauts, therefore, it is critical to monitor the physiological indicators with the purpose to study the damage mechanisms and means of protection. As a special space environments, microgravity can lead to mitochondrial dysfunction. Since mitochondrial membrane potential is an important indicator of normal mitochondria, as a result, 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. 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 approach to investigate the cells under microgravity environment.