A colorimetric and fluorescent probe C-Py-1 for ONOO(-) was prepared and it could exhibit high sensitivity and excellent selectivity toward ONOO(-) among reactive oxygen species (ROS) and reactive nitrogen species (RNS) with a rapid response time in 100% water solution. Meanwhile, C-Py-1 was successfully applied in the imaging of endogenous ONOO(-) in RAW264.7 cells.