H2S is an essential gas signal molecule in cells, and viscosity is a key internal environmental parameter. Recent studies have shown that H2S acts as a cytoarchitecture agent and gas transmitter in many tissues, e.g., as a regulator of neuroendocrine in the brain for mediating vascular tone in blood vessels. Mitochondrial viscosity is an important parameter for judging whether mitochondrial function is normal. It has been reported that oxidative stress and mitochondrial dysfunction are connected with Parkinson’s disease (PD), and the protective role of H2S in PD models has been extensively demonstrated. Herein, Mito-HS, a new two-photon fluorescent probe was demonstrated to detect cross-talk between the two channels of mitochondrial viscosity and H2S content. Moreover, this probe could detect the relative amount of and changes in mitochondrial H2S in situ due to the reduced mitochondrial targeting ability after reaction with H2S. The results show that H2S in mitochondria is inversely related to viscosity. The PD model has a lower H2S in mitochondria and a higher mitochondrial viscosity than did the normal. This result is important for our deep understanding of PD and its causes.