Hydrogen peroxide (H₂O₂) as a crucial signal molecule plays a vital part in the growth and development of various cells under normal physiological conditions. The development of H₂O₂ sensors has received great research interest because of the importance of H₂O₂ in biological systems and its practical applications in other fields. In this study, a H₂O₂ electrochemical sensor was constructed based on chalcogenide molybdenum disulfide–gold–silver nanocomposite (MoS₂-Au-Ag). Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and energy dispersive spectroscopy (EDS) were utilized to characterize the nanocomposites, and the electrochemical performances of the obtained sensor were assessed by two electrochemical detection methods: cyclic voltammetry and chronoamperometry. The results showed that the MoS₂-Au-Ag-modified glassy carbon electrode (GCE) has higher sensitivity (405.24 µA mM⁻¹ cm⁻²), wider linear detection range (0.05–20 mM) and satisfactory repeatability and stability. Moreover, the prepared sensor was able to detect the H₂O₂ discharge from living tumor cells. Therefore, this study offers a platform for the early diagnosis of cancer and other applications in the fields of biology and biomedicine.