The ideal dermal matrix should be able to provide the right physical and biological environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the biocompatible interactions with tissue. Chitosan (CS)/poloxamer semi-interpenetrating polymer networks (SIPNs) was prepared by crosslinking of poloxamer macromer in the presence of CS in order to improve its mechanical property required in skin tissue engineering application. Furthermore, we also prepared CS/poloxamer SIPNs with ShebaTM, human acellular dermis, to overcome a low biocompatibility of the CS/poloxamer SIPNs. The CS/poloxamer SIPNs/ShebaTM showed remarkably highly cell attachment and viability in the two-dimensional (2D) culture and similar cell morphology on the CS/poloxamer SIPNs/ShebaTM compared with on tissue culturing polystylene (TCPS) as a positive control. These results suggest that CS/poloxamer SIPNs containing ShebaTM have good possibility for artificial skin system application.