A microfiber platform that is able to enhance neuronal differentiation and guide aligned neurite outgrowths is essential to the repair of nerve damage. To achieve this aim, we utilized biocompatible and biodegradable poly lactic-co-glycolic acid (PLGA) to design a novel Aligned Contiguous Microfiber Platform (ACMFP) as substrates for the neuronal induction of mouse embryonic stem (ES) cells. To generate the ACMFP, a modified micro-fluid chip system was established to control microfiber parameters including fiber diameter, alignment, and the distance between fibers. Further, Pluronic-F127 was applied to the ACMFP system to maintain a stable and highly aligned fiber platform for at least 12 days. We found that the ACMFP can enhance the neuronal differentiation of mouse ES cells. The ACMFP system showed significantly better neurite outgrowth alignment guidance compared to the control substrate. The effects of alignment guidance were inversely proportionate to the diameter of the fiber, with the optimal diameter size of 60 µm. This study demonstrates a novel ACMFP system that can be used as a biomaterial substrate for neurite outgrowth alignment guidance, which may provide a new model for the development of a multidisciplinary treatment option for nerve injuries.