Cardiac surgery and cardiopulmonary bypass (CPB) are associated with a systemic inflammatory reaction that occasionally induces a life-threatening organ dysfunction caused by the dysregulated host response to the damage-associated molecular patterns (DAMPs). In severe inflammation, cell-free DNA (cfDNA) and histones are released by inflammatory cells and damaged tissue and act as DAMPs. We sought to characterize the changes in circulating cell-free DNA (cfDNA) levels during CPB. Primary outcomes were renal failure, ventilation time (>18 hr), length of stay (LOS) in the intensive care unit (ICU) (>48hr), hospital LOS (>15 days), and death. We looked for associations with blood tests and comparison to standard scores. In a prospective cohort study, we enrolled 71 patients undergoing non-emergent coronary artery bypass grafting. Blood was drawn at baseline, 20 and 40 minutes on CPB, after cross-clamp removal, and 30 minutes after chest closure. cfDNA was measured by our fast fluorescent method. Baseline cfDNA levels [796 (656-1063) ng/ml] increased during surgery, peaked after cross-clamp removal [2403 (1981-3357) ng/ml] and returned to baseline at recovery. The difference in cfDNA from 20 to 40 minutes on CPB (ΔcfDNA 40-20) inversely correlated with peripheral vascular disease (PVD), longer ventilation time, and longer ICU and hospital length of stay (LOS). Receiver operating characteristic (ROC) curve of ΔcfDNA 40-20 for long ICU-LOS (>48hr) was with an area under the curve (AUC) of 0.738 (p = 0.022). ROC AUC of ΔcfDNA 40-20 to long Hospital LOS (>15 days) was 0.787 (p = 0.006). Correction for time on CPB in a multivariate logistic regression model improved ROC-AUC to 0.854 (p = 0.003) and suggests that ΔcfDNA 40-20 is an independent risk factor. To conclude, of measured parameters, including STS and Euroscore, the predictive power of ΔcfDNA 40-20 was the highest. Thus, measurement of ΔcfDNA 40-20 may enable early monitoring of patients at higher risk. Further studies on the mechanism behind the negative association of ΔcfDNA 40-20 with PVD and outcomes are warranted.