It is important to know the mechanism of cell membrane fluctuation because it can be readout for the nanomechanical interaction between cytoskeleton and plasma membrane. Traditional techniques, however, have drawbacks such as probe contact with the cell surface, complicate analysis, and limit spatial and temporal resolution. In this study, we developed a new system for non-contact measurement of nano-scale localized-cell surface dynamics using modified-scanning ion-conductance microscopy. With 2 nm resolution, we determined that endothelial cells have local membrane fluctuations of ~20 nm, actin depolymerization causes increase in fluctuation amplitude, and ATP depletion abolishes all membrane fluctuations.