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Response Surface Methodology (RSM) was used to assess the optimal conditions for a Water/Oil/Water (W/O/W) emulsion for encapsulated nisin (EN). Nano-encapsulated nisin had high encapsulation efficiencies (EE) (86.66 ± 1.59%), small particle size (320 ± 20 nm), and low polydispersity index (0.27). Biodegradable polyvinyl alcohol (PVA) and polyacrylate sodium (PAAS) were blended with EN and prepared by electrospinning. Scanning electron microscopy (SEM) revealed PVA/PAAS/EN nanofibers with good morphology, and that their EN activity and mechanical properties were enhanced. When the ultrasonication time was 15 min and 15% EN was added, the nanofibers had optimal mechanical, light transmittance, and barrier properties. Besides, the release behavior of nisin from the nanofibers fit the Korsemeyer–Peppas (KP) model, a maximum nisin release rate of 85.28 ± 2.38% was achieved over 16 days. At 4 °C, the growth of Escherichia coli and Staphylococcus aureus was inhibited for 16 days in nanofibers under different ultrasonic times. The application of the fiber in food packaging can effectively inhibit the activity of food microorganisms and prolong the shelf life of strawberries, displaying a great potential application for food preservation.