In this study, PEUU elastomer was synthesized and nanofibers with five different PEUU/gelatin ratios (PEUU/gelatin = 0 : 100, 25 : 75, 50 : 50, 75 : 25, 100 : 0, which were called PU0, PU25, PU50, PU75, PU100, respectively) were fabricated via electrospinning. All samples were treated in glutaraldehyde (GA treating) for 24 h. The SEM image results show that there is a uniform distribution for all the nanofibers and the mean fiber diameters increase with the increase in the percentage of PEUU. The FTIR spectra indicate that PEUU was successfully synthesized. The XRD results show that the GA treating process decreased the crystallinity of PEUU to some extent. It can be concluded from the mechanical results that the mechanical properties of PU100 and PU75 nanofibers are superior to other groups. Only a small amount of gelatin significantly improved the hydrophilic properties of the nanofibers. The results of cell proliferation and cell morphology indicate that the PEUU/gelatin nanofibers, particularly the PU75 group, could better contribute to the cell proliferation than the PU0 and PU100 groups, supporting the application of PU75 in skin tissue engineering. Two different types of electrospun nanoyarns were also fabricated, which showed better comprehensive properties than nanofiber, thus paving way for further research in the tissue engineering field.