The absence of an effective imaging tool for diagnosing renal ischemia-reperfusion injury (RIRI) severely delays its treatment, and currently, no definitive clinical interventions are available. Pyroglutamate aminopeptidase-1 (PGP-1), a potential inflammatory cytokine, has shown considerable potential as a biomarker for tracing the inflammatory process in vivo. However, its exact role in the enhanced visualization of RIRI in complex biological systems has yet to be fully established. Chemiluminescence imaging (CLI) has proven to be one of the most promising diagnostic methods due to its ultrahigh-contrast imaging capabilities compared to fluorescence imaging. In this study, we developed an activatable Schaap's dioxetane chemiluminescent probe (PGP-PD) to explore the potential of PGP-1 as a marker for CLI of renal injury following ischemia-reperfusion, with the goal of achieving high-contrast in situ diagnostics for RIRI. In vitro, PGP-PD exhibited exceptional selectivity for exogenous PGP-1 and remarkable sensitivity, with a detection limit as low as 2.244 ng/mL. Moreover, in vivo studies successfully demonstrated a positive correlation between the RIRI and PGP-1 level. Notably, in situ imaging with PGP-PD generated a significant chemiluminescent signal within the RIRI-kidney, providing an exceptionally high contrast between injured and normal kidney tissue (∼9.4-fold) in the RIRI mouse model. We anticipate that this work may offer a valuable biomarker (PGP-1) and a powerful imaging tool for improving RIRI in situ diagnosis, thereby aiding treatment planning and surgical outcomes for RIRI patients.