Sepsis is a life-threatening infectious disease. Mitochondrial dysfunction is widespread in severe sepsis. The myocardium contains a large number of mitochondria, and the survival rate of sepsis decreases sharply when cardiac dysfunction is involved. Vericiguat (BAY 1021189) is a novel drug for the prevention of heart failure. In this study, we evaluated the mitochondrial function of septic mice and drug-treated mice by resonance Raman spectroscopy (RRS). RRS is a non-invasive and label-free technique that can identify molecular vibrations by their unique fingerprints, making it ideal for quantitative studies. By choosing 532 nm as the excitation wavelength, which is close to the absorption peak of cytochrome, we can greatly enhance the Raman signal of mitochondrial redox state. RRS can accurately identify the Raman characteristic peak at 750 cm−1, 1128 cm−1 and 1585 cm−1 attributed to the reduced cytochrome in septic mice. We found that the intensity of the characteristic peak was significantly decreased in septic mice, indicating an imbalance of mitochondrial redox function, while the function was improved in the drug-treated group. It proves that BAY has the potential as a novel treatment for mitochondrial dysfunction in sepsis.