ONOO- and SO2 are important microenvironment parameters in organisms, the intracellular levels of ONOO- and SO2 are abnormal in rheumatoid arthritis (RA) mice. Mitophagy plays a key role in maintaining mitochondrial quality and quantity, and viscosity monitoring is a crucial aspect of real-time visualization of mitophagy. Therefore, a mitochondria-targeting three-channel fluorescent probe HCPVP was first developed for the detection of ONOO-/SO2 and viscosity. HCPVP can sensitively detect ONOO- (514 nm) and SO2 (674 nm) change with different channels, meanwhile display turn-on fluorescence at 678 nm as the amount of glycerol increased. The fluorescence imaging of cells showed that HCPVP was able to better target mitochondria, monitor ONOO-/SO2 changes, and visualize the mitophagy process. HCPVP has been successfully used for monitoring ONOO- and SO2 in RA mice model, providing the early image of RA with the aid of the potential biomarkers ONOO- and SO2. More importantly, HCPVP could evaluate the response of the RA treatment with impressive results, which held the potential for assessing treatment and drug screening for RA.