In vitro studies have been popularly used to determine the cellular and molecular mechanisms for many decades. However, the traditional two-dimension (2D) cell culture which grows cells on a flat surface does not fully recapitulate the pathological phenotypes. Alternatively, the three-dimension (3D) cell culture provides cell-cell and cell-ECM interaction that better mimics tissue-like structure. Thus, it has gained increasing attention recently. Yet, the expenses, time-consuming, and complications of cellular and biomolecular analysis are still major limitations of 3D culture. Herein, we describe a cost-effective and simplified workflow of the 3D neuronal cell-laden agarose-laminin preparation and the isolation of cells, RNAs, and proteins from the scaffold. To study the effects of the amyloidogenic condition in neurons, we utilized a neuron-like cell line, SH-SY5Y, and induced the amyloidogenic condition by using an amyloid forty-two inducer (Aftin-4). The effectiveness of RNAs, proteins and cells isolation from 3D scaffold enables us to investigate the cellular and molecular mechanisms underlying amyloidogenic cascade in neuronal cells. The results show that SH-SY5Y cultured in agarose-laminin scaffold differentiated to a mature TUJ1-expressing neuron cell on day 7. Furthermore, the gene expression profile from the Aftin-4-induced amyloidogenic condition revealed the expression of relevant gene-encoding proteins in the amyloidogenic pathway, including APP, BACE1, PS1, and PS2. This platform could induce the amyloid-beta 42 secretion and entrap secreted proteins in the scaffold. The induction of amyloidogenic conditions in a 3D culture facilitates the interaction between secreted amyloid-beta and neurons, which makes it resembles the pathological environment in Alzheimer's brain. Together, this workflow is applicable for studying the cellular and molecular analysis of amyloid-induced neuronal toxicity, such as those occurred in Alzheimer's disease progression. Importantly, our method is cost-effective, reproducible, and easy to manipulate.