Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.