Regenerated silk fibroin (RSF) is a promising candidate for skin repair due to its wound healing properties. RSF films have some drawbacks such as weak mechanical property, low porosity and poor absorption which limits their application as an ideal wound healing dressing. In this study, a novel RSF-based nanohybrid bearing carnosine (Car) intercalated bentonite (CarBen) nanosheets was prepared using solvent casting method and was applied in a second-degree burn wound model. RSF bearing different amounts of CarBen nanosheets (0, 1, 3 and 5 wt%) was prepared and fully characterized. RSF having 5 wt% of the modified Ben (RSF-CarBen5) showed superior mechanical property (ultimate stress of 8.36 ± 01 MPa and ultimate strain of 1501.33 ± 1.24%), and higher absorption capacity (1653.57 ± 0.05%) with favorable porosity (80.27%). RSF-CarBen5 was employed as dressing for healing of second-degree burn wound in rats. Histological evaluation showed considerable inflammation reduction in wound sites compared to the control groups. The wound treated with the nanohybrid showed significant epithelialization and angiogenesis on day 14 post-treatment, compared to the other groups (P < 0.05). The results showed that RSF bearing CarBen nanosheets have suitable therapeutic potential and can heal burn wounds appropriately.