Fibrous chitosan membranes were fabricated as a substrate for skin applications using an electro-spinning process with different solvents and varying concentrations. Scanning electron microscopy (SEM) images confirmed that the formation of the chitosan fibrous membrane in trifluoroacetic acid was better than that in acetic acid. Fourier transform infrared spectroscopy showed that the chitosan fibers were cross-linked with glutaraldehyde, and that the cytotoxicity of the aldehyde groups was reduced by glycine and washing by NaOH and DI water. Chitosan cross-linked fibrous membranes were insoluble in water and could be washed thoroughly to wash away glycine and excess NaOH and prevent the infiltration of other water soluble bio-toxic agents using DI water. MTT assay method was employed to test the cytotoxicity of chitosan membranes during fabricating, treating and washing processes. After the dehydration of cell cultured chitosan membranes, cell attachment behavior on the material was evaluated using SEM method. Effect of the treatment processes on the biocompatibility of the chitosan membranes was shown by comparing of filopodium and lamellipodium of fibroblast cells on grown washed and unwashed chitosan fibrous membrane. The MTT assay and SEM morphology confirmed that the washed chitosan fibrous membrane increased cell attachment and cell growth, and decreased toxicity compared to results for the unwashed chitosan fibrous membrane.