The cochlear basilar membrane (CBM) contains inner hair cells and outer hair cells that convert sound waves into electrical signals and transmit them to the central auditory system. Cochlear aging, the primary reason of age-related hearing loss, can reduce the signal transmission capacity. There is no ideal in vitro aging model of the CBM. In this study, we cultured the CBM, which was dissected from the cochlea of the C57BL/6 mice 5 days after birth, in a medium containing 20 mg/mL, 40 mg/mL, or 60 mg/mL D-galactose (D-gal). Compared with the control group, the levels of senescence-associated β-galactosidase were increased in a concentration-dependent manner in the CBM of the D-gal groups. In addition, levels of the mitochondrial superoxide and patterns of an age-related mitochondrial DNA3860-bp deletion were significantly increased. The ATP levels and the membrane potential of the mitochondrial were significantly decreased in the CBM of the D-gal groups compared with the control group. Furthermore, in comparison with the control group, damaged hair cell stereocilia and a loss of inner hair cell ribbon synapses were observed in the CBM of the D-gal groups. A loss of hair cells and activation of caspase-3-mediated outer hair cell apoptosis were also observed in the CBM of the high-dose D-gal group. These insults induced by D-gal in the CBM in vitro were similar to the ones that occur in cochlear natural aging in vivo. Thus, we believe that this is a successful in vitro aging model using cultured CBM. These results demonstrate the effects of mitochondrial oxidative damage on presbycusis and provide a reliable aging model to study the mechanisms of presbycusis in vitro.