In situ monitoring of cellular metabolism is useful for elucidating dynamic functions of living cells. In our previous studies, cellular respiration was continuously monitored as a change in pH at the cell/electrode nanoscale interface (i.e., interfacial pH) using an ion-sensitive field-effect transistor (ISFET). However, such interfacial pH behaviour on the nanoscale has not been confirmed using other methods such as fluorescence imaging. In this study, we have clarified the interfacial pH behaviour at a cell/substrate nanogap using a laser scanning confocal fluorescence microscope. The phospholipid fluorescein used as a pH indicator was fixed to the plasma membrane on the external side of a cell by inserting its lipophilic alkyl chain into the membrane, and used to observe the change in interfacial pH. As a result, hydrogen ions generated by cellular respiration were gradually accumulated at the cell/substrate nanogap, resulting in a decrease in pH. Moreover, the interfacial pH between the plasma membrane and the substrate became lower than the pH near the surface of cells not in contact with the substrate. The data obtained in this study support the idea that potentiometric ion sensors such as ISFETs can detect a cellular-metabolism-induced change in pH at a cell/electrode nanogap in real time.