Cell-based neural regeneration is challenging due to the difficulty in obtaining sufficient neural stem cells with clinical applicability. Stem cells from apical papilla (SCAPs) originating from embryonic neural crests with high neurogenic potential could be a promising cell source for neural regeneration. This study aimed to investigate whether the formation of 3D spheres can promote SCAPs’ neurogenic potential. Material and methods: Three-dimensional SCAP spheres were first generated in a 256-well agarose microtissue mold. The spheres and single cells were individually cultured on collagen I-coated μ-slides. Cell morphological changes, neural marker expression, and neurite outgrowth were evaluated by confocal microscope, ELISA, and RT-qPCR. Results: Pronounced morphological changes were noticed in a time-dependent manner. The migrating cells’ morphology changed from fibroblast-like cells to neuron-like cells. Compared to the 2D culture, neurite length, number, and the expression of multiple progenitors, immature and mature neural markers were significantly higher in the 3D spheres. BDNF and NGF-β may play a significant role in the neural differentiation of SCAP spheres. Conclusion: The formation of 3D spheres enhanced the neurogenic potential of SCAPs, suggesting the advantage of using the 3D spheres of SCAPs for treating neural diseases.