In the present study, we developed a novel approach for functionalization of gelatin nanofibers using the plasma method for tissue engineering applications. For this purpose, tannic acid-crosslinked gelatin nanofibers were fabricated with electrospinning, followed by treatment with argon and argon–oxygen plasmas in a vacuum chamber. Samples were evaluated by using scanning electron microscopy (SEM), atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle (CA) and X-ray diffraction (XRD). The biological activity of plasma treated gelatin nanofibers were further investigated by using fibroblasts as cell models. SEM studies showed that the average diameter and the surface morphology of nanofibers did not change after plasma treatment. However, the mean surface roughness (RMS) of samples were increased due to plasma activation. ATR-FTIR spectroscopy demonstrated several new bands on plasma treated fibers related to the plasma ionization of nanofibers. The CA test results stated that the surface of nanofibers became completely hydrophilic after argon–oxygen plasma treatment. Finally, increasing the polarity of crosslinked gelatin after plasma treatment resulted in an increase of the number of fibroblast cells. Overall, results expressed that our developed method could open new insights into the application of the plasma process for functionalization of biomedical scaffolds. Moreover, the cooperative interplay between gelatin biomaterials and argon/argon–oxygen plasmas discovered a key composition showing promising biocompatibility towards biological cells. Therefore, we strongly recommend plasma surface modification of nanofiber scaffolds as a pretreatment process for tissue engineering applications.