Superoxide (O2•-) is a specific molecular target for xenobiotics, cytokines, and bacteria during inflammatory diseases. The aim of this study is to develop a single-photon emission computed tomography (SPECT) imaging agent and to quantify the distribution of O2•- in vivo. 125/131I-PISO was obtained in good radiochemical yield (65.4 ± 9.2%) and high radiochemical purity (>98%) after HPLC purification. 125/131I-PISO (log P = 2.46) could be oxidized by O2•- selectively and sensitively, converted to a hydrophilic compound 125/131I-PISA (log P = -1.62) with negative charge simultaneously and conglutinated with biomolecules by electrostatic interactions. The specific accumulation of 131I-PISA in the O2•- rich region were verified in cell efflux assay and SPECT/CT imaging in situ O2•- enrichment model mice. SPECT/CT imaging showed higher accumulation of 125I-PISO in the inflamed ankles compared to the control. Radioiodinated PISO is a potential SPECT agent to image O2•- distribution in vivo through specific and sensitive O2•- triggered retention.