Neurite outgrowth is the critical step of nervous development. Molecular probes against neurites are essential for evaluation of the nervous system development, compound neurotoxicity, and drug efficacy on nerve regeneration. To obtain a neurite probe, we developed a neurite-SELEX strategy and generated a DNA aptamer, yly12, that strongly binds neurites. The molecular target of yly12 was identified to be neural cell adhesion molecule L1 (L1CAM), a surface antigen expressed in normal nervous system and various cancers. Here, yly12 was successfully applied to image the three-dimensional network of neurites between live cells, as well as the neurite fibers on normal brain tissue section. This aptamer was also found to have an inhibitory effect on neurite outgrowth between cells. Given the advantages of aptamers, yly12 hold great potential as a molecular tool in the field of neuroscientific research. The high efficiency of neurite-SELEX suggests that SELEX against a subcellular structure instead of the whole cells is more effective in obtaining the desired aptamers.