A multicellular tumor aggregate, known as a spheroid, is an indispensable tool to study cancer biology. Owing to its three-dimensional organization, a spheroid exhibits an inherent gradient of nutrients, oxygen, and metabolites within itself. The spheroid provides culture conditions that resemble the microenvironment of certain cancer cells and causes these cells to acquire characteristics relevant to tumors in our body. However, site-specific gene expression analysis in an intact spheroid with single-cell resolution has not been explored. Recently, some types of electrochemical syringes were developed to extract cellular materials from living single cells for transcriptomic analysis. Here, we investigated whether an electrochemical syringe could be used to evaluate site-specific gene expression in a spheroid. A small amount of cytosol (roughly 540-1480 fL, less than the volume of a single cell) was successfully collected from the first, second, and third layers of the spheroid using an electrochemical syringe without causing damage to the spheroid architecture. We found that the CCNB1 and CCNA2 expression levels were different between the surface and the average of the entire spheroid, indicating that there are heterogeneous cellular functions across different regions of the spheroid. This method provides opportunities to improve our understanding of spatial gene expression of single cells in a three-dimensional environment.