A novel chitosan-based membrane that made of hydroxyethyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane including transparency, equilibrium water content, ion and glucose permeability were determined. The results showed that the optical transparency of the membrane was as good as the natural human cornea. The water content of this scaffold was 81.32% which was remarkably close to the native cornea. The membrane had a good ion permeability and its glucose permeability was even higher than natural human cornea. The cultured rabbit corneal endothelial cells formed a monolayer on the membrane. The results demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow on it. In addition, the membranes in vivo could be degraded steadily with less inflammation and showed a good histocompatibility. These results demonstrated that the hydroxyethyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.