Promotion of neurite outgrowth is an important limiting step for regeneration in nerve injury and depends strongly on the local expression of nerve growth factor (NGF). The rational design of bioactive materials is a promising approach for the development of novel therapeutic methods for nerve regeneration, and biomaterials capable of presenting NGF to nerve cells are especially suitable for this purpose. In this study, we show bioactive peptide amphiphile (PA) nanofibers capable of promoting neurite outgrowth by displaying high density binding epitopes for NGF. A high-affinity NGF-binding sequence was identified by phage display and combined with a beta-sheet forming motif to produce a self-assembling PA molecule. The bioactive nanofiber had higher affinity for NGF compared to control nanofibers and in vitro studies revealed that the NGF binding peptide amphiphile nanofibers (NGFB-PA nanofiber) significantly promote the neurite outgrowth of PC-12 cells. In addition, the nanofibers induced differentiation of PC-12 cells into neuron-like cells by enhancing NGF/high-activity NGF receptor (TrkA) interactions and activating MAPK pathway elements. The NGFB-PA nanofiber was further shown as a promising material to support axonal outgrowth from primary sensory neurons. These materials will pave the way for the development of new therapeutic agents for peripheral nervous system injuries.