In this study, a scanning electrochemical microscope (SECM) was for the first time adapted to perform the electroporation process of living yeast cells. We have demonstrated that relatively low voltage pulses of 1–2 V vs. Ag/AglCl,Cl−sat applied to gold-based ultramicroelectrode (Au-UME) are performing reversible electroporation of yeast cells immobilized on fluorine-doped tin oxide (FTO)/glass surface. SECM and electrochemical impedance spectroscopy (EIS) were used for the determination of quantitative electrochemical characteristics before and after the electroporation. The electrochemical impedance spectroscopy (EIS) illustrated significant electrochemical changes of electroporated yeast cells, while SECM feedback mode surface vertical scan current-distance curves showed that the diameter of the area affected by the electrical pulse is about 25 times larger than the diameter of the Au-UME used for the electroporation process. The results presented in this research open up a possibility to develop a targeted electroporation system which will affect only the selected area of tissue or some other cell-covered surface. Such model is promising for the selective treatment of selected cells in tissues and/or other sensitive biological systems while selecting the location and size of electroporated areas.