Dihydrorhodamine 123 (DHR 123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydrorhodamine 6G (DHR 6G) were evaluated as probes for detecting cellular hydrogen peroxide levels in SPC-A-1 lung adenocarcinoma cells. Imaging techniques and fluorescence-activated cell scan were used in the study of the probe responses. Obvious green fluorescence was established after a 25-min exposure. After staining with MitoTracker Orange CM-H2TMRos (a probe for mitochondria) and the abovementioned probes simultaneously, only the DHR 123 and DHR 6G groups exhibited legible green fluorescence in the mitochondrial regions. Furthermore, the DHR 6G group exhibited weaker fluorescence intensity. When 100 μM H2O2 was added to SPC-A-1 cells loaded with these probes, the intracellular fluorescence increased rapidly and significantly. Our results suggest that DHR 123 is superior for the instantaneous detection of cellular hydrogen peroxide in SPC-A-1 cells.