Objective To explore the value of serum matrix metalloproteinase-9 (MMP-9) and cystatin-C (Cys-C) in prediction of acute kidney injury (AKI) in the elderly. Methods A total of 116 AKI patients in our ward from June 2016 to June 2018 were enrolled as AKI group. Another 68 patients without AKI during the same period were subjected as non-AKI group. Fasting peripheral venous blood samples were collected to detect the serum levels of MMP-9 and Cys-C by enzyme-linked immunosorbent assay (ELISA). The levels were compared between 2 groups. Receiver operating characteristic (ROC) curve analysis was used to analyze the value of MMP-9 and Cys-C in predicting AKI individually and jointly. SPSS statistics 20.0 was used for data analysis. Results The serum levels of MMP-9 [(377.61±28.72) vs (96.50±20.42)ng/ml] and Cys-C [(3.22±0.76) vs (0.92±0.16)mg/L] were significantly higher in the AKI group than in the non-AKI group (P<0.05). The area under ROC curve (AUC) of MMP-9 in predicting AKI was 0.714, the Yoden index was the highest when the cut-off point was 190ng/ml, and the sensitivity and specificity were 77% and 63%, respectively. Cys-C predicted AKI with an AUC of 0.805 and cut-off point of 2.0mg/L, and the Yoden index was the highest with sensitivity of 80% and specificity of 75%. The AUC of AKI predicted by their combination was 0.856, sensitivity was 85%, and specificity was 81%, which was significantly higher than those of individual markers (P<0.05). Conclusion The serum levels of MMP-9 and Cys-C are elevated in elderly AKI patients. The sensitivity and specificity of combined prediction of AKI by MMP-9 and Cys-C are higher than those of single marker of MMP-9 or Cys-C.