Polyelectrolyte complex (PEC) membrane of cationic chitosan and anionic sodium alginate with fiber structure were prepared by freeze-drying method. Chitosan and sodium alginate were blended in different concentrations and frozen at different temperatures. Freeze-dried fiber membranes were extensively characterized for their inter-molecular interaction, the solution property, morphology and biocompatibility by using FTIR, XRD, zeta (ζ) potentials, SEM and cytotoxicity assay. The study of swelling property showed that PEC membrane with the fiber structure cross-linked with glutaraldehyde exhibited pH and ionic strength-dependent swelling in aqueous media, which might have a potential application in tissue engineering or drug controlled release. Furthermore, chitosan–sodium alginate samples showed better cell adhesion and proliferation than pure chitosan. The results indicated that two natural polyelectrolyte complex nanofibers were prepared by freeze-drying method and fitted for tissue engineering or as drug carriers.