The SH-SY5Y neuroblastoma cell line is a widely used in vitro model for studying neuronal differentiation and neurodegenerative conditions such as Alzheimer’s disease (AD) due to its ability to undergo neuronal-like differentiation and express key neuronal markers relevant to AD pathology. However, current differentiation protocols often lack physiological relevance, primarily due to the absence of extracellular matrix (ECM) components critical for mechanotransduction and cellular adhesion. This study describes a novel and efficient method for differentiating SH-SY5Y cells into neuron-like cells by combining retinoic acid (RA) treatment with a laminin-rich ECM (LrECM). Our approach significantly enhanced neuronal differentiation markers, including β3-tubulin and NeuN, and promoted extensive neurite outgrowth. Immunofluorescence analysis showed increased expression of these markers, confirming enhanced neuronal characteristics. Western blot results further validated sustained β3-tubulin expression over time. Adding LrECM also augmented cholinergic differentiation, as evidenced by increased acetylcholinesterase activity measured through colorimetric assays. The findings highlight the critical role of LrECM in enhancing neuronal differentiation and maintaining neuron-like characteristics over longer periods. This approach provides a valuable improvement for refining in vitro models used in the study of neuronal development and neurodegenerative diseases, particularly AD.