Extracellular H₂O₂ released from single COS-7 cells was quantitatively and spatially detected using scanning electrochemical microscopy (SECM). For the first time, depth scan imaging approach in vertical x-z plane was applied to a single cell for obtaining probe approach curves (PACs) to any position on the membrane of the cell in the image by simply drawing a vertical line on the depth SECM image. This novel SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The concentration of H₂O₂ at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O₂, and determined to be 0.020 mM by overlapping the experimental PAC with simulated one. The H₂O₂ profile by means of SECM gives insight into physiological activity of single live cells. In addition, intracellular H₂O₂ profile was demonstrated using confocal microscopy by labelling the cells with 2’,7’-dichlorodihydrofluorescein diacetate. The two techniques showed complementary experimental results of H₂O₂ detection, demonstrating that H₂O₂ generation is centered at endoplasmic reticula.