Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.