Abstract: Viscosity is an essential micro-environmental parameter and hydrogen sulfide (H₂S) is a critical gaseous signaling molecule, which are both related to various physiological and pathological processes. It has practical significance to detect the concentration of H₂S and viscosity change. In this work, we developed a fluorescent probe Mito-ANP for simultaneous detecting H₂S and viscosity. After the increase of viscosity, the rotation of the probe is prohibited and an intense red fluorescence is released. While, upon the addition of H₂S, the C = C of the probe can react with H₂S via the nucleophilic addition to form Mito-ANP-H ₂ S and a strong green fluorescence can be observed. Moreover, the probe Mito-ANP exhibited excellent relationship between viscosity and H₂S. In addition, the probe Mito-ANP was successfully to used for fluorescent imaging of viscosity and H₂S changes in neurocyte cells.