Abstract Current diagnostics for sepsis‐associated acute kidney injury (SA‐AKI) detect kidney damage only at advanced stages, limiting opportunities for timely intervention. A DNA origami‐based nanoplatform is developed for the early diagnosis and treatment of SA‐AKI. Modified with a fluorophore (Cy5) and quencher (BHQ3), the DNA origami remains nonfluorescent under normal conditions. During SA‐AKI, elevated microRNA‐21 triggers a strand displacement reaction that restores the fluorescence signal, enabling real‐time detection. Additionally, the photoacoustic changes of BHQ3, driven by different excretion rates of the nanostructure and released DNA strands, enable dual‐mode imaging, enhancing diagnostic accuracy. Therapeutically, DNA origami scavenges reactive oxygen species and, when conjugated with the antimicrobial peptide Leucine‐Leucine‐37 (LL‐37), exhibits bactericidal effects. This combination boosts survival rates by 80% in SA‐AKI models. This dual‐response nanoplatform integrates precise imaging and targeted therapy, offering a powerful strategy for SA‐AKI management and advancing applications of DNA origami in precision nanomedicine.