Currently, autologous nerve (AN) transplantation remains the gold standard for treating peripheral nerve injuries (PNIs). However, its inherent limitations, including donor site morbidity and immune rejection risks associated with allografts, have prompted the exploration of alternative therapeutic strategies. Among these, tissue engineering approaches have gained significant attention, with nerve conduit design emerging as a particularly promising research direction. Electrospinning technology has been widely adopted for its ability to fabricate nanofibrous scaffolds that closely mimic the native extracellular matrix. In this study, we engineered an aligned nanofiber conduit utilizing polylactic acid and gelatin through electrospinning, and integrated a sodium alginate hydrogel enriched with Schwann cells (SCs) and neural stem cells (NSCs) via coaxial bioprinting. The three-dimensional (3D) hydrogel microenvironment facilitated synergistic interactions between SCs and NSCs, augmenting the secretion of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). A dynamic perfusion culture system was further employed to optimize cell viability and functionality. In vivo studies revealed that the implantation of this conduit in a sciatic nerve defect model markedly enhanced motor function recovery, nerve regeneration, and muscle morphology. These improvements were substantiated by an increased sciatic functional index (SFI), heightened expression of S-100 and NF-200, and greater myelin thickness and axon diameter. Although the efficacy of the 3D-aligned nanofiber conduit cocultured with SCs and NSCs approximated that of AN transplantation, further research is imperative to identify more efficient seed cells and biocompatible 3D carriers to achieve optimal nerve regeneration. This study highlights the potential of tissue-engineered nerve conduits as a viable alternative for PNI repair, paving the way for future advancements in the field.