The development of advanced wound dressings capable of accelerating healing and achieving effective hemostasis remains a critical challenge in managing traumatic and surgical wounds. This study reports the fabrication and comprehensive evaluation of a novel multilayered, sandwich-structured nanofiber scaffold composed of Tranexamic acid (TXA), chitosan (CS), polyvinyl alcohol (PVA), L-arginine, and polylactic acid (PLA) for biodegradable wound dressing applications. Using sequential electrospinning, a four-layered scaffold was developed as a smart wound dressing, comprising an immediate-release TXA-CS-PVA layer for rapid clotting, a hydrophobic PLA barrier layer for protection, a sustained-release L-arginine-PVA layer to promote tissue regeneration, and a final PLA protective layer to enhance durability. The morphology and fiber alignment were optimized by employing both flat plate and rotating disc collectors to achieve random and aligned nanofiber structures, respectively. Characterization studies confirmed successful Tranexamic acid (TXA) encapsulation and structural integrity of the scaffolds. In vitro cytotoxicity tests showed excellent biocompatibility, while in vivo full-thickness wound models demonstrated superior wound closure rates, enhanced collagen deposition, and elevated TGF-β1 expression with minimal skin irritation. Notably, scaffolds with aligned fibers and thinner PLA barriers significantly accelerated healing compared to random fibers and marketed formulations. These findings highlight the promising potential of the fabricated multilayered electrospun nanofiber scaffold as an effective biodegradable wound dressing that not only accelerates hemostasis but also promotes enhanced wound healing.