Abstract Accurate early diagnosis and prognosis of sepsis remain major clinical challenges. This study explores specific plasma cell‐free mitochondrial DNA (cfmtDNA) breakage patterns as potential biomarkers for sepsis. Plasma samples from ten non‐sepsis control patients and 63 sepsis patients are analyzed using mitochondrial DNA fragmentomics, revealing distinct breakage sites in the RNR2 (positions 2474–2478) and COX2 (positions 7761, 7775, 7776, 7777, and 7783) regions, which are intact in healthy individuals but exhibited high‐frequency breakage in sepsis patients. Diagnostic models based on these breakage sites show superior accuracy for sepsis detection (AUC = 0.865) and prognosis prediction (AUC = 0.809) compared to traditional cfmtDNA copy number assessments. Notably, COX2 breakage frequency correlated with inflammatory markers and SOFA scores, highlighting its prognostic potential. Mechanistic analyses suggest that reduced protein binding in sepsis may increase cfmtDNA susceptibility to cleavage by bacterial restriction endonucleases. These findings indicate that plasma cfmtDNA breakage characteristics can serve as valuable biomarkers for early sepsis detection and therapeutic monitoring.