Fluorescence imaging has emerged as an attractive technology for the real-time and noninvasive detection of biomarkers in cellular and animal-based applications. Previously, several single-analyte fluorescent probes have been reported for the selective imaging of ONOO -. Moreover, fluorescent probes for the selective detection and visualization of ATP have been developed. However, to date no fluorescent probe capable of the simultaneous and independent detection and imaging of ONOO -and ATP has been reported, such a system would allow us to monitor this close relationship in real time. Here, we report the construction of ATP-LW, a single fluorescent probe that enables the simultaneous detection of ONOO -and ATP.