Silk fibroin (SF) scaffolds have been widely used in tissue engineering. However, a critical challenge for 3D SF scaffolds remains in providing a more appropriate microenvironment with a nanofibrous network to enhance cell viability and guide cell migration, thus further promoting tissue regeneration. In this study, a novel SF scaffold containing micro-/nano-fibers was prepared by a facile two-step freeze-drying technology. Carbodiimide-activated SF solution was diluted to 0.2 wt%, and then poured into pre-fabricated porous SF scaffolds. Consequently, well-dispersed fibrous networks with a fiber size of 511 ± 217 nm were produced within the pores of SF scaffolds after liquid nitrogen immersion, followed by lyophilization. The results of in vitro culture of dermal fibroblast cells and umbilical vein endothelial cells on fibrous SF scaffolds demonstrated that the introduction of the micro-/nano-fibers significantly enhanced cell attachment, proliferation and migration by providing 3D topographic cues. In vivo, the SF scaffolds were implanted into dorsal full-thickness wounds of Sprague-Dawley rats as dermal equivalents to evaluate the effect of the fibrous microstructure on dermal tissue reconstruction. The results demonstrated that the fibrous SF scaffolds promoted tissue neogenesis and collagen matrix formation by providing a fibrous ECM-like topography. This new fibrous SF scaffold offers potential for dermal tissue regeneration.