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Purpose : The purpose of present study was to develop a bi-layer membrane.Type Ⅰ collagen/chitosan/poly(ethylene oxide)nanofibrous film with porous allowing cells adhesion and proliferation;Collagen/chitosan film was on the opposite surface with freeze-dried for inhibition cells invasion.Methods: Chitosan or collagen dissolved in 1%(v/v)HAc solutions to prepare a 2%and 0.5%(w/v)solution,respectively.After removing entrapped air-bubbles,the mixed solution frozen at-20℃、-40℃、-80℃ for several hours,lyophilized for 24h to obtain a porous membrane.