Abstract Background Developing biocompatible nerve conduits that accelerate peripheral nerve regeneration, lengthening and functional recovery remains a challenge. The combined application of nerve microtissues and platelet-rich plasma (PRP) provides abundant Schwann cells (SCs) and various natural growth factors and can compensate for the deficiency of SCs in the nerve bridge, as well as the limitations of applying a single type of growth factor. Multimodal ultrasound evaluation can provide additional information on the stiffness and microvascular flow perfusion of the tissue. This study was designed to investigate the effectiveness of a novel tissue-engineered nerve graft composed of an autogenous vein, nerve microtissues and PRP in reconstructing a 12-mm tibial nerve defect and to explore the value of multimodal ultrasound techniques in evaluating the prognosis of nerve repair. Methods In vitro, nerve microtissue activity was first investigated, and the effects on SC proliferation, migration, factor secretion, and axonal regeneration of dorsal root ganglia (DRG) were evaluated by coculture with nerve microtissues and PRP. In vivo, seventy-five rabbits were equally and randomly divided into Hollow, PRP, Micro-T (Microtissues), Micro-T + PRP and Autograft groups. By analysing the neurological function, electrophysiological recovery, and the comparative results of multimodal ultrasound and histological evaluation, we investigated the effect of these new nerve grafts in repairing tibial nerve defects. Results Our results showed that the combined application of nerve microtissues and PRP could significantly promote the proliferation, secretion and migration of SCs and the regeneration of axons in the early stage. The Micro-T + PRP group and Autograft groups exhibited the best nerve repair 12 weeks postoperatively. In addition, the changes in target tissue stiffness and microvascular perfusion on multimodal ultrasound (shear wave elastography; contrast-enhanced ultrasonography; Angio PlaneWave UltrasenSitive, AngioPLUS) were significantly correlated with the histological results, such as collagen area percentage and VEGF expression, respectively. Conclusion Our novel tissue-engineered nerve graft shows excellent efficacy in repairing 12-mm defects of the tibial nerve in rabbits. Moreover, multimodal ultrasound may provide a clinical reference for prognosis by quantitatively evaluating the stiffness and microvescular flow of nerve grafts and targeted muscles.