Neuroblastoma (NB) is a solid pediatric malignancy of the sympathetic nervous system, characterized by pronounced phenotypic heterogeneity and a complex tumor microenvironment (TME). In this context, extracellular vesicles (EVs), small membrane-enclosed particles naturally secreted by cells, are emerging as key mediators of intercellular communication implicated in tumor progression, immune evasion, and metastatic dissemination; however, their specificity across distinct NB cell populations remains poorly understood, and dedicated platforms to investigate EV targeting in pediatric cancer are still lacking. Therefore, a stepwise tissue-engineering approach was implemented to develop biomimetic NB models of increasing complexity for the investigation of EV targeting dynamics. Initially, a three-dimensional porous collagen type I/hyaluronic acid (Col1/HA) scaffold was employed to produce NB-derived EVs, given its capacity to recapitulate the architecture and biophysical properties of native NB tissue.