Compared with normal cells, cancer or tumor cells have a specific microenvironment and apparently possess a relatively large amount of ROS/RNS, and their overexpression is one of the important reasons for tumor development and deterioration. Therefore, monitoring the changes of intracellular ROS/RNS can improve the awareness of the clinical manifestations of the disease, which will be beneficial for the early diagnosis of cancer and improving treatment efficiency. Herein, in this study we have exploited and constructed a novel strategy based on the [email protected] nanowire electrode for intracellular electrochemical analysis to monitor ROS levels in cancer or tumor cells. Firstly, the [email protected] nanowire electrode was utilized to detect the intracellular ROS radical changes involved in the relevant biological processes of cancer cells where fluorescent zinc nanoclusters were biosynthesized in situ in target cancer cells by using the intracellular microenvironment and specificity of these cancer cells. By combining a confocal fluorescence microscopy study simultaneously, our observations illustrate that accompanied by the apparent change of the intracellular ROS, these in situ biosynthesized fluorescent nanoclusters gradually accumulate inside the cytosolic area with the increase of the reaction time. Moreover, it is evident that the size of the [email protected] nanoelectrodes can match the single cell dimensions, and its unique high spatial resolution provides the possibility of relevant intracellular molecular detection. These nanoelectrochemical biosensors can be adopted to quantitatively determine the change of the ROS content in target single cells in the relevant biological microenvironment or during the in situ biosynthesis process, and are also beneficial for understanding the related mechanism of some specific biological processes including the in situ synthesis at the single cell level.