Background: Due to the lack of diagnostic efficiency of serum creatinine in acute kidney injury (AKI), there is a pressing need to develop novel diagnostic markers. Therefore, in this study, we evaluated myo-inositol oxygenase (MIOX), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C in terms of their applicability in the diagnosis of AKI. Methods: We enrolled a total of 39 AKI patients and 38 healthy controls in the study. We compared the levels of serum MIOX, NGAL and cystatin C between the two groups. Results: We found that the concentrations of serum creatinine, blood-urea nitrogen, MIOX and cystatin C were higher in the AKI group. According to the receiver operating characteristic analysis, the area under the curve (AUC) values were 0.694 (95% CI 0.579-0.794) for MIOX and 0.976 (95% CI; 0.912-0.997) for cystatin C. For MIOX, when the cut-off concentration was set to 77.3 pg/mL, the diagnostic sensitivity and specificity were found to be 53.8% (95% CI; 37.2-69.9) and 81.5 (95% CI; 65.7-92.3), respectively. For cystatin C, at the cut-off value of 14 mg/L, the diagnostic sensitivity and specificity were 94.8% (95% CI; 82.7-99.4) and 94.7 % (95% CI 82.3-99.4), respectively. Conclusion: The measurement of serum MIOX and cystatin C levels is valuable for the diagnosis of AKI. Further research is needed for the evaluation of the potential use of MIOX as a kidney-specific enzyme in the early diagnosis of AKI.