Distinguishing between normal, inflammatory, and progressing tumor cells plays a vital role in early diagnoses and clinical studies. The simultaneous quantification of multiple biomarkers in cells can reveal cellular heterogeneity, which contributes to the discrimination of different types of cells. Herein, a dual-channel fluorescent probe has been developed for monitoring peroxynitrite (ONOO-) and glutathione (GSH) to accurately discriminate normal cells, inflammatory cells, and progressing cancer cells. The probe can monitor exogenous and endogenous mitochondrial GSH and ONOO- in living cells and zebrafish by green (530 nm, G530) and red (630 nm, R630) emission based on its good selectivity and low biotoxicity. GSH and ONOO- are visualized via fluorescence imaging, and the corresponding output signals can be employed to differentiate nontumorigenic, malignant, and metastatic breast cells in cocultured cells. Furthermore, the accurate discrimination among normal, inflammatory, and cancerous cells is achieved through the changes in the dual-channel fluorescence signal, which shows great potential for the diagnosis of inflammation and cancer diseases.