Adenosine triphosphate (ATP) is a direct energy source in cells and the core of the biochemical system, and is closely related to various metabolic activities in living organisms. Therefore, designing a simple and rapid ATP detection method is significant to study its physiological function. Herein, a dual-channel fluorescent probe RhB-NA for the in situ imaging of ATP in living cells was designed and synthesized. When ATP bound to RhB-NA, the spirolactam in rhodamine B was induced to open, resulting in a new fluorescence response at 589 nm. Notably, in cell imaging, the treatment of HeLa cells with exogenous H2O2 and H2S, which have certain effects on the mitochondria, confirmed that RhB-NA could detect fluctuations in ATP levels after the mitochondrial state was affected. We believe that RhB-NA has far-reaching significance for studying certain physiological diseases caused by abnormal ATP levels.