We developed the three-dimensional (3D) hydrogel-based microfluidic device to study the shear stress and tumor migration. Gelatin methacrylate (GelMA) hydrogel was used to culture the human umbilical vein endothelial cells (HUVECs) in a 3D microfluidic device. We investigated the effect of shear stress on HUVEC alignments in the circular-shaped microfluidic channels. It showed that the HUVECs exposed to shear stress for 2 days in the circular-shaped microfluidic channel tended to elongate and align with fluidic directions in a controlled manner. We also observed that the tumors migrated into GelMA hydrogel containing vascular endothelial growth factor (VEGF). Therefore, this hydrogel-based 3D microfluidic device could be a powerful method for studying the shear stress and tumor migration applications.