Objective To investigate the clinical value of peripheral serum cell-free DNA/neutrophil extracellular traps (cf-DNA/NETs) level in diagnosis and severity assessment of sepsis patients. Methods Forty patients with sepsis and 40 patients with non-infectious systemic inflammatory response syndrome (nf-SIRS) were enrolled in this study. The cf-DNA/NETs level in serum of all subjects were measured. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic ability of the cf-DNA/NETs, white blood cell count (WBC), procalcitonin (PCT) and interleukin-6 (IL-6). The sepsis patients were stratified into a survival group and a death group according to the prognosis. Sequential organ failure (SOFA) score were recorded in the sepsis patients, and the correlations between SOFA and cf-DNA/NETs, PCT, WBC, IL-6 were analyzed. Results Compared with the nf-SIRS group, cf-DNA/NETs and PCT levels were significantly higher in the sepsis group (both P<0.05). WBC and IL-6 showed no significant differences between the two groups (bothP>0.05). The area under the ROC curve (AUC) of cf-DNA/NETs was 0.884 for diagnosis of sepsis, and it was higher than the AUC of PCT (0.803). The cf-DNA/NETs showed better sensitivity (81.2% and 79.2%) and specificity (81.0% and 82.4%) than PCT. cf-DNA/NETs and PCT were significantly higher in the death group than those in the survival group. Bivariate collection analysis revealed positive correlations between SOFA score and the two biomarkers of cf-DNA/NETs and PCT (r1=0.573, r2=0.518; both P<0.01). Conclusions cf-DNA/NETs and PCT have certain value in early diagnosis of sepsis, and cf-DNA/NETs shows better diagnostic value in distinguishing sepsis from nf-SIRS than PCT. cf-DNA/NETs can be used as a routine monitoring index to help assess disease severity in sepsis.