Myocardial fibrosis is frequently accompanied by elevated levels of oxidative stress. Mitochondrial glutathione (mGSH), an essential biomolecule for maintaining redox homeostasis in mitochondria, could serve as an effective indicator for investigating the oxidative stress associated with myocardial fibrosis. In this study, a ratiometric fluorescent probe named Mito-NS6, capable of being anchored in mitochondria and reversibly responding to GSH with an appropriate dissociation equilibrium constant, was rationally designed and utilized to visualize and evaluate the changes of mGSH levels caused by oxidative stress in myocardial fibrosis. Benefiting from the good performance of Mito-NS6, we successfully achieved the quantification of mGSH in cardiac fibroblasts using a confocal laser-scanning microscope, revealing that salvianolic acid B (SalB) can act as an effective drug to alleviate myocardial fibrosis through depressing oxidative stress. Moreover, we employed ratio fluorescence imaging to track the fluctuation in GSH levels within a mice model of myocardial fibrosis induced by isoproterenol and found that myocardial fibrosis caused a higher oxidative stress level in myocardial tissue as well as heart organs. These results provide a novel point of view for the diagnosis and treatment of myocardial fibrosis.