Traumatic nerve defects result in dysfunctions of sensory and motor nerves and are usually accompanied by pain. Nerve guidance conduits (NGCs) are widely applied to bridge large-gap nerve defects. However, few NGCs can truly replace autologous nerve grafts to achieve comprehensive neural regeneration and function recovery. Herein, a three-dimensional (3D) sponge-filled nanofibrous NGC (sf@NGC) resembling the structure of native peripheral nerves was developed. The conduit was fabricated by electrospinning a poly(L-lactide-co-glycolide) (PLGA) membrane, whereas the intraluminal filler was obtained by freeze-drying a collagen-based matrix (ColM) resembling the extracellular matrix. The effects of the electrospinning process and of the composition of ColM on the physicochemical performance of sf@NGC were investigated in detail. Furthermore, the biocompatibility of the PLGA sheath and ColM were evaluated. The continuous and homogeneous PLGA nanofiber membrane had high porosity and tensile strength. ColM was shown to exhibit an ECM-like architecture characterized by a multistage pore structure and a high porosity level of over 70%. The PLGA sheath and ColM were shown to possess stagewise degradability and good biocompatibility. In conclusion, sf@NGC may have a favorable potential for the treatment of nerve reconstruction.