Gelatin nanofibers (GNs) prepared by electrospinning were cross-linked with glutaraldehyde vapor to improve their water-resistant ability. After cross-linking treatment, the form of the fibers expressed no substantial change, but the average diameter of the fibers increased with increasing cross-linking time. The swelling induced by the moisture during the cross-linking process was moderated when the cross-linking time reached 45 min. The contact angle measurements confirmed that the electrospun gelatin fibers were more hydrophilic than the gelatin film (GF). Increasing the cross-linking time did not alter the hydrophilic properties of the gelatin fibers. The cell compatibility was evaluated based on 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope and confocal microscope observations, and Western blot analysis by culturing MG-63 cells on the GFs and GNs. The nanofibrous structure fabricated by an electrospinning technique was found to enhance cell adhesion and proliferation. This process is a cost-effective simulation of GN structures' promising applications on scaffold preparation for tissue engineering.