Monitoring the fluctuation of adenosine 5'-triphosphate (ATP) at the subcellular level is important for the study of cell energy metabolism. Herein, we fabricated an electrochemical nanoaptasensor for continuously monitoring ATP fluctuation at the subcellular level. A gold nanoelectrode with a diameter of 120 nm was fabricated, and ferrocene (Fc)-labeled anti-ATP aptamer was self-assembled onto the nanoelectrode surface to form a nanoaptasensor. In the presence of ATP, the ferrocene-labeled anti-ATP aptamer bound with two ATP units to form an ATP-aptamer conjugation, resulting in the close proximity of Fc to the nanoelectrode surface and then an increase of oxidation current of Fc. ATP can be detected with a detection limit of 26 μM within 2 min. Cell viability assays indicated that the nanoaptasensor was biocompatible with negligible biological effects. By taking advantage of the good biocompatibility of the nanoaptasensor, ATP fluctuation at the subcellular level was monitored under glucose starvation and Ca2+ induction. This work demonstrates that the nanoaptasensor is a useful tool for investigating ATP-relevant biological processes via the electrochemical method.