Biochemical and physical guidance cues are both pivotal for axonal guidance and nerve regeneration. However, fabrication of a platform that can integrate biochemical gradients and topographical guidance cues remains challenging, especially in a three-dimensional (3D) scaffold that closely mimics in vivo axonal outgrowth conditions and ready to be used for in vivo nerve repair. In this study, a new method was introduced to construct 3D scaffolds displaying continuous biochemical gradients along longitudinally oriented microchannels by combining the modified 3D printing and directional freezing techniques. Fluorescence analysis and ELISA results demonstrated that a continuous biochemical gradient was formed, and scanning electron microscopy revealed a longitudinally oriented microstructure. Dorsal root ganglia explants seeded on the longitudinal sections of the newly developed scaffold (scaffold with nerve growth factor gradient along oriented microstructure, G-NGF + OS) showed that 81.3 ± 4.5% of neurites oriented within ±10°, 0.3 ± 0.1 of guidance ratio, and 1.5-fold of the average length of neurites on the high-nerve growth factor (NGF) concentration side compared to that on the low-NGF concentration side, which were significantly higher than those in the other groups. In addition, the G-NGF + OS scaffold was used to repair a 15 mm sciatic nerve defect in rats. Immunofluorescence staining, Fluoro-Gold retrograde tracing, and transmission electron microscopy results confirmed that the G-NGF + OS scaffold enhanced nerve regeneration to the distal target and promoted myelination of regenerated axons. More importantly, the sciatic functional index and the von Frey test demonstrated that the G-NGF + OS scaffold accelerated sensory and motor functional recovery. These results provide new insights into the importance of combining topographical guidance cues with bioactive molecule gradient cues for neural tissue engineering. The 3D scaffold displaying biochemical gradients along longitudinally oriented microchannels represents a promising platform for the development of advanced devices for severe nervous system injuries.