Objective To evaluate the clinical significance of serum Klotho protein levels in the early diagnosis and prognosis of acute kidney injury (AKI) among adult patients in the intensive care units (ICU). Methods The study was prospective and observational. Blood samples and clinical data of AKI patients admitted to the ICU of the First Affiliated Hospital of Xinjiang Medical University between July 1 and August 31, 2016 were collected. ELISA was used for the detection of Klotho and NGAL. Receiver operating characteristic curve (ROC) and the area under the curve (AUC) were used to compare the predictive performance among Klotho, NGAL and serum creatinine, evaluating the sensitivity and specificity of Klotho on the diagnosis of AKI. The correlation between Klotho and prognosis of AKI was investigated by comparing serum Klotho levels and early AKI predictors. Results The patients were divided into AKI group of 52 cases and non-AKI group of 98 cases. The baseline serum Klotho level in AKI group was significantly lower than that in non-AKI group (P＜0.001). The AUC of Klotho predicting for AKI was 0.945(95% CI: 0.892-0.997) and the best cut off value was 1.76 μg/L(sensitivity 92%, specificity 94%). The predictive ability of Klotho was significantly higher than serum creatinine (Scr), and the sensitivity is higher than NGAL (sensitivity 87%, specificity 96%). Serum Klotho combined with Scr predicted better AKI (AUC=0.958, 95% CI: 0.915-1.000, sensitivity 96%, sensitivity 92%). The level of Klotho in patients with AKI was significantly different between the renal function recovery group and non-recovery group (P=0.047), while there was no significant difference between the two groups in the level of NGAL and Scr (P＞0.05). There was no significant correlation between the Klotho level at diagnosis of AKI and peak Scr, peak eGFR, Scr at discharge and eGFR at discharge (r=0.026, P=0.853; r=-0.127, P=0.368; r=0.243, P=0.082; r=-0.187, P=0.184). Conclusion Serum Klotho may be a potential biomarker for early diagnosis of AKI, but the association between serum klotho and the prognosis of AKI requires further study.