In this study, polylactic acid (PLA)/sulfadiazine sodium/proteinase K composite nanofibers were prepared by electrospinning, and their morphology was observed by scanning electron microscope. The novel nanofibers were characterized by Fourier transform infrared spectroscopy and drug release curve. The activity of different groups of drugs loaded nanofibers in frostbite wound of rats was studied and compared with the blank control group. Hematoxylin-eosin staining and transmission electron microscope were used to access the histological changes of frostbite wounds on the back of rats before and after treatment.CD31 fluorescence staining was used to track the reconstruction and improvement of blood vessels in tissues. The results revealed that the drug release rate of PLA/sulfadiazine sodium/proteinase K nanofibers group was more than 50% at 1 h, and the cumulative drug release rate was more than 88% at 168 h, and CD31 fluorescence showed that its vascular density was 13.27% ± 0.9%, which was significantly higher than that of other groups. Therefore, PLA/sulfadiazine sodium/proteinase K composite nanofibers could promote the healing of frostbite wounds, which indicated the increase of capillaries and the regeneration of hair follicles. PLA/sulfadiazine sodium/proteinase K composite nanofibers have great potential in the application of frostbite.