Gelatin nanofibers have gained significant attention for different biomedical applications, as they provide a suitable environment for cell attachment, growth, and proliferation compared to the other biopolymers and synthetic polymers. Airbrushing/solution-blow-spinning could overcome the limitation of the conventional electrospinning method of nanofiber preparation. The present study reports the fabrication of nano/microfibers from commercially available low-molecular-weight gelatin of animal origin as a first-time study. The effect of various airbrushing parameters, namely, the concentration of gelatin solution, air pressure, and polymer solution flow rate on the fiber quality, morphology, and diameters, was studied. Finally, the biological evaluation of the airbrushed gelatin nanofibers was performed using human bone marrow-derived mesenchymal stem cells (hBMSCs). Gelatin nanofibers exhibit excellent biocompatibility and support the growth of hBMSCs similar to electrospun gelatin nanofibers. Our airbrushing technique is an easy, low-cost, and scalable method to fabricate the gelatin nanofibers for different biomedical applications such as tissue engineering, wound healing, and substrate for delivery of bioactive molecules. © 2019 American Chemical Society.