This work aimed to prepare pH-sensitive nanofibrous mats as a drug-releasing system using a green method. Gelatin nanofibers were first prepared by electrospinning and then cross-linked with glutaraldehyde. To evaluate the capability of this product as a drug delivery system, vancomycin was loaded into the nanofibrous mats in different doses as a model antibiotic drug. The chemical structure of the prepared material was investigated by (Fourier transform-infrared) FT-IR. Field emission scanning electron microscopy (FE-SEM) observations showed that uniform bead-free nanofibers with an average diameter of 157 nm were successfully fabricated. The drug release studies revealed that the relative rate of drug release in buffer media with pH =2.0 was higher than that in a buffer solution with pH =7.4. The drug release mechanism of samples was determined by the Korsmir-Pepas model. Moreover, the incorporation of vancomycin into the nanofibers provided an effective antibacterial activity against Escherichia coli and Staphylococcus aureus microorganisms. The developed antibiotic-loaded nanofibrous mats can be considered a promising novel antimicrobial wound dressing material.