Despite significant developments in optical imaging of superoxide anion (O-2(center dot-)) as the preliminary reactive oxygen species, novel visualizing strategies that offer ultrahigh sensitivity are still imperative. This is mainly because intrinsic concentrations of O2(center dot-) are extremely low in living systems. Herein, we present the rational design and construction of a new polymer nanoprobe PCLA-O-2(center dot-) for detecting O-2(center dot-) based on chemiluminescence (CL) resonance energy transfer without an external excitation source. Structurally, PCLA-O-2(center dot-) contains two moieties linked covalently, namely imidazopyrazinone that is capable of CL triggered by O-2(center dot-) as the energy donor and conjugated polymers with light-amplifying property as the energy acceptor. Experiment results demonstrate that PCLA-O-2(center dot-) exhibits ultrahigh sensitivity at the picomole level, dramatically prolonged luminescence time, specificity, and excellent biocompatibility. Without exogenous stimulation, this probe for the first time in situ visualizes O-2(center dot-) level differences between normal and tumor tissues of mice. These exceptional features ensure that PCLA-O-2(center dot-). as a self-luminescing probe is an alternative in vivo imaging approach for ultralow level O-2(center dot-).