Mitochondrial dysfunction and oxidative stress are key contributors to age-related hearing loss (ARHL). Sestrin2, a stress-inducible antioxidant protein, decreases with age, impacting various age-related diseases; however, its role in ARHL remains unclear. In this study, we initially assessed hearing in 8-week-old mice, followed by adeno-associated virus-mediated transfection of Sestrin2 into the posterior semicircular canal. At 26 weeks, hearing was reassessed, and cochlear samples were analysed. Immunofluorescence staining revealed Sestrin2 localization and mitophagy marker PINK1 expression, and TUNEL staining was used to assess hair cell apoptosis rate. We also measured mitochondrial membrane potential and examined AMPKα, mTOR, PINK1, Parkin, and Sestrin2 protein levels. Results showed reduced Sestrin2 in ARHL-affected hair cells, leading to mitochondrial dysfunction and increased apoptosis. Sestrin2 overexpression enhanced mitochondrial membrane potential, ATP levels, and mitochondrial function, delaying hair cell ageing and apoptosis and improving hearing. Furthermore, AMPK activation inhibited mTOR to promote mitophagy as the cells aged, whereas Sestrin2 overexpression inhibited this pathway, directly protecting hair cells. These findings highlight Sestrin2’s critical role in auditory health and its potential as a therapeutic target for delaying ARHL.