A novel nisin-loaded poly (vinyl alcohol)/wheat gluten/zirconia (Nisin-PVA/WG/ZrO2) nanofibrous membranes was prepared via electrospinning technique. The drug carrier PVA/WG/ZrO2 nanofibers showed a increase in average diameter with the increase of ZrO2 content, and the fibers tended to conglutinate when ZrO2 content reached 12 wt% and above. Hydrogen and Zr-O-C bonds existed among PVA, WG, and ZrO2 units improved the thermal stability of polymeric matrix. The loading dosage of nisin caused no significant changes in the size and morphology of nanofibers at nisin content below 6 wt%. The antimicrobial activity and release experimental results showed that the nisin-PVA/WG/ZrO2 nanofibrous membranes displayed well-controlled release and better antimicrobial activity against Staphylococcus aureus, and the nisin release from the nanofibers could be described by Fickian diffusion model. In our opinions, these nisin-PVA/WG/ZrO2 nanofibrous membranes may have potential as a new nanofibrous membrane in drug delivery, wound dressing, and active food packaging.