Cellular senescence is one of the barriers to maintain in vitro three-dimensional (3D) epidermal models for a long period of time. Therefore, a new culture substrate should be developed to suppress keratinocyte senescence to establish an epidermal model. In this study, reconstituted extracellular matrices (ECM) were prepared by culturing keratinocytes at different passages using the decellularization technique. The ECM prepared by decellularization (dECM) supports keratinocyte adhesion and growth. It has also been demonstrated that the dECM suppresses keratinocyte senescence by increasing the antioxidant activity. In particular, the dECM derived from younger passaged keratinocytes suppresses senescence more significantly than the dECM derived from highly passaged keratinocytes. Moreover, the dECM derived from younger passaged keratinocytes can suppress keratinocyte senescence during passage culture. Finally, the dECM derived from younger passaged keratinocytes increased AQP3 gene expression as an indicator of the functions of basal keratinocytes and the AQP3 expression ability to respond to all-trans retinoic acid. The dECM derived from younger passaged keratinocytes could be a useful culture substrate for developing an in vitro epidermal model.