This study developed multi-layered lidocaine/epinephrine/collagen loaded biodegradable nanofibrous scaffolds and evaluated their efficacy for pain relief and hemostasis in palatal oral wounds of rabbits. Twelve rabbits were enrolled and randomly divided into two groups. Free gingival grafts were first harvested from palatal mucosa. The wounds in the test group were dressed by multi-layered biodegradable nanofibrous scaffolds, while the wounds were exposed in the control group. Daily changes of body weight, and food/water intakes were recorded. Histological examinations were also completed. All scaffolds exhibited good biocompatibility and eluted effective levels of lidocaine and epinephrine at the initial stages of wound recovery. Rabbits in the test group showed faster hemostasis as well as recovery of food and water intake postoperatively, compared with those in the control group. The multi-layered biodegradable nanofibrous scaffolds offered adequate efficacy of hemostasis and sustainable pain relief for the initial healing in rabbits with palatal oral wounds.