The current study aimed to develop a potential wound dressing using vitamin B12-loaded polycaprolacton/gelatin nanofibrous scaffold. In order to produce wound dressings, 1000 mcg of vitamin B12 was added to polycaprolacton/gelatin solution and the nanofibrous scaffolds were fabricated through electrospinning method. The obtained scaffolds were studied regarding their hydrophobicity, microstructure, amount of water absorption, water vapor permeability, tensile strength, release test, and cellular proliferation assay. In vitro studies revealed that the incorporation of vitamin b12 into polycaprolacton/gelatin scaffolds could significantly augment L929 cells proliferation at 1 and 3 days post-seeding. However, there was not statistically significant difference between Vitamin B12-containing and polymer-only scaffolds in tensile strength study, surface wettability measurement, water vapor transmission test, the capacity for water absorption, and nanofiber's diameter. Both vitamin containing and free dressings were applied on the full-thickness excisional wound in rat model to compare their healing potential. Our results showed that after 14 days, vitamin B12 containing dressing could significantly enhance wound closure compared to vitamin B12 free scaffolds (92.27 ± 6.84% vs. 64.62 ± 2.96%). Furthermore, histopathological examinations showed significantly greater epithelial thickness in polycaprolacton/gelatin/vitamin B12 group compared to other experimental groups. This preliminary study suggest potential applicability of the proposed dressing to treat skin wounds in clinic.