Recent trends in peripheral nerve regeneration are directed toward the development of nerve guidance channels to assist the regeneration of the nerves across critical size defects. Advanced nerve guidance channels (aNGCs) should possess multifunctional properties to direct the axonal regeneration from proximal to distal end, allow the concentration of growth factors secreted by the injured nerve end, and attenuate the ingrowth of scar tissue at the site of injury. The design of the nerve guidance channel (NGC) is critical for providing the necessary topographical, chemotactic, as well as haptotactic cues for efficient nerve regeneration. In this study, we have designed and fabricated clinically relevant aNGCs comprising an antioxidant polyurethane (PUAO) conduit filled with aligned chitosan-gelatin (CG) cryogel filler for peripheral nerve regeneration. The effects of temperature, polymer concentration, and cross-linker concentration on the physicochemical properties of the CG cryogel filler were studied. The synthesized scaffolds were evaluated by scanning electron microscopy (SEM) and compression testing to obtain the matrix best suited to form the aNGC. The nanofibrous PUAO conduit was fabricated by electrospinning with a wall thickness of 114.16 ± 26.91 μm, which was filled with CG (1.2/6.4%)-aligned cryogel matrix to obtain the aNGCs. The aNGCs with 2.01 ± 0.04 mm internal diameter, 15 mm length, and internal CG filler with a pore diameter of 29.60 ± 9.83 μm were fabricated. The aNGCs were evaluated by SEM and in vitro neuronal culture for biocompatibility and cellular alignment. In vitro dorsal root ganglion cultures showed the aligned growth and cellular migration along the aligned pores of aNGCs. With this study, we conclude that this clinically relevant aligned porous aNGC will have a promising effect in repair and regeneration of peripheral nerve injuries.