Peroxynitrite (ONOO-) is one type of important reactive oxygen/nitrogen species (ROS/RNS) and plays a vital role in many physiological activities. Excessive ONOO- is associated with many diseases including inflammation, arthritis, inflammatory bowel disease, cancer, and neurodegenerative diseases. However, a chemiluminescent probe capable of detecting endogenous ONOO- at the acidic condition that might be applied for sensitive diagnosis of inflammation-related disease has not been reported. Hence, we designed and synthesized a chemiluminescence (CL) probe, B-PD, to detect endogenous ONOO- both in vitro and in vivo. B-PD demonstrated a quick response toward ONOO- with a limit of detection of 201 nM. In vivo CL imaging results showed that, 30 min postinjection, B-PD could effectively locate early stage inflammation tissue with an imaging contrast up to 6.2. These results suggest that B-PD holds great promise for highly sensitive diagnosis of inflammation-related diseases in the future.