The gelatin-glutaraldehyde (gelatin-GA) nanofibers were electrospun in order to overcome the defects of ex-situ crosslinking process such as complex process, destruction of fiber morphology and decrease of porosity. The morphological structure, porosity, thermal property, moisture absorption and moisture retention performance, hydrolytic resistance, mechanical property and biocompatibility of nanofiber scaffolds were tested and characterized. The gelatin-GA nanofiber has nice uniform diameter and more than 80% porosity. The hydrolytic resistance and mechanical property of the gelatin-GA nanofiber scaffolds are greatly improved compared with that of gelatin nanofibers. The contact angle, moisture absorption, hydrolysis resistance, thermal resistance and mechanical property of gelatin-GA nanofiber scaffolds could be adjustable by varying the gelatin solution concentration and GA content. The gelatin-GA nanofibers had excellent properties, which are expected to be an ideal scaffold for biomedical and tissue engineering applications.