Cellular dynamics is very closely related to ionic behaviors, most of which have been hardly monitored in real time, whereas semiconductor-based biosensors have the unique advantage of direct detection of ionic charges in a real-time and noninvasive manner. In this study, we monitored the invasion process of cancer cells into the vascular endothelial layer in real time by a label-free method using a field-effect transistor (FET) biosensor. Endothelial cells were cultured on the sensing surface of the FET gate, to form a basement membrane between the endothelial cells and the sensing surface. When invasive cancer cells (HeLa cells) approached the endothelial cell-coated gate FET biosensor, a change in the surface potential was clearly detected using the FET biosensor. This is because HeLa cells, which invaded the endothelial cell layer, reduced the molecular charge density in the basement membrane by decomposing it. A platform based on the cell-coated gate FET biosensor is suitable for real-time and noninvasive monitoring of cellular dynamics based on intrinsic ionic charges.