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In this paper, Poly (diallyldimethylammonium chloride) (PDDA)/honey nanofiber wound dressing composites were prepared and their effects on the diabetic wound-healing was evaluated using in vivo experiments. The release of effective compounds and the solubility of nanofibers were controlled through the crosslinking process by glutaraldehyde. The crosslinked nanofibers (crosslinking time was 3 h) showed an absorption capacity at a maximum value of 989.54%. Interestingly, the resultant composites were able to prevent 99.9% of Staphylococcus aureus and Escherichia coli bacteria. Furthermore, effective compounds were continuously released from nanofibers for up to 125 h. In vivo evaluation indicated that the use of PDDA/honey (40/60) significantly enhanced wound-healing. On the day 14th, the average healing rate for samples covered by conventional gauze bandage, PDDA, PDDA/honey (50/50), and PDDA/honey (40/60) were 46.8 ± 0.2, 59.4 ± 0.1, 81.7 ± 0.3, and 94.3 ± 0.2, respectively. The prepared nanofibers accelerated the wound-healing process and reduced the acute and chronic inflammation. Hence, our PDDA/honey wound dressing composites open up new future treatment options for diabetic wound diseases.