Sepsis-associated acute kidney injury (SA-AKI) severely impacts morbidity and mortality in surgical patients with sepsis. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) have an important role in pathophysiology of sepsis but they have been unexplored in SA-AKI. We aimed to investigate the role of MMP-9 and TIMP-1 in septic surgical patients with SA-AKI and to evaluate them as diagnostic biomarkers of SA-AKI. This prospective observational study compared 53 major abdominal surgery patients with sepsis divided into SA-AKI (n= 37) and non-SA-AKI (n= 16) group to 50 controls without sepsis matched by age, gender, comorbidities and type of surgery. Blood and urine samples from septic patients were collected on admission to ICU and 24, 48, 72 and 96 h later and once from the controls. The levels of MMP-9, TIMP-1, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1, urea and creatinine were measured. MMP-9/TIMP-1 ratio and disease severity scores, such as Sequential Organ Failure Assessment (SOFA), were calculated. Septic patients with SA-AKI had higher serum TIMP-1 levels and lower serum MMP-9 levels and lower MMP-9/TIMP ratio, compared to septic patients without SA-AKI and controls. The levels of these biomarkers did not change significantly over time. MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio correlated with urea, creatinine, NGAL, and SOFA scores. Moreover, using the area under ROC curve, we showed that TIMP-1 and MMP-9/TIMP-1 ratio, but not MMP-9, were good diagnostic biomarkers of SA-AKI. We report for the first time the potential diagnostic value of TIMP-1 and MMP-9/TIMP-1 ratio in SA-AKI.