Glioblastoma (GBM) is the most aggressive and malignant brain tumor with no effective treatment available so far. A major obstacle in GBM research is the lack of reliable in vitro models that can mimic the tumor cellular environment. The goal of this study was to develop a 3D-engineered scaffold as a new cell culture model capable of providing a more accurate representation of the tumor environment. 3D-engineered scaffolds, inspired by the geometry of brain blood vessels, were fabricated using two-photon polymerization. A newly developed biocompatible material with low auto-florescence was used for fabrication. To assess the ability of the 3D model in mimicking GBM’s natural environment, cell colonization, cellular morphology, microtube formations, and epidermal growth factor receptor (EGFR) expressions of cells were derived from confocal microscopy images and compared with those in 2D control experiments. The EGFR expressions were further evaluated in the presence of two EGFR inhibitors. U-87 cell line and three primary GBM cell cultures successfully colonized the scaffolds. Morphological differences were observed between 2D and 3D models with 3D models more closely representing in vivo observations. Microtube-like protrusions were present in both 2D and 3D models but the 3D model better matched the morphology of microtubes in vivo. There were no significant differences in expressions and subcellular localizations of EGFR between 2D and 3D models. However, EGFR internalizations were not prevented in the presence of EGFR inhibitors. This previously-unreported observation may explain the in vivo resistance of GBM cells to inhibitors. The 3D-engineered scaffolds provided a new suitable model that better replicated the GBM’s natural environment with advantages that were difficult to capture in 2D models.