Abstract: Objective To investigate the bioactivity in the in vitro cultured tissue engineering skin. Methods Epithelial cells and fibroblasts were seeded onto the surface of an acellular dermal matrix to form a skin substitute, and the culture supernatants were collected on the 3rd and the 7th day. The changes in the content of interleukin-6 and interleukin-8 (IL-6,8) and transforming growth factor 1 (TGF-β1) in the supernatants were measured by enzyme linked immunosorbent assay ( ELISA), while the changes in the content of laminin, hyaluronic acid, type Ⅰ and type Ⅲ collagen were quantified by radioactive immunoassay (RIA). Results A certain amounts of cytokines, growth factors and extracellular matrix could be detected in the supernatant of the skin substitute, and the contents of these substances on the 7th day were significantly higher than those on the 3rd day( P <0.01) . The contents of type I and type Ⅲ collagen in the skin substitute containing both epidermis and fibroblast were dramatically lower compared with those in the skin substitute only containing fibroblast( P <0.01). Conclusion The in vitro cultured skin substitute possessed relatively powerful bioactivity, and the fibroblast function could be modulated to some extent by seeding epidermis on it.