Background: Patients with acute myocardial infarction (AMI) are at high risk for acute kidney injury (AKI). Novel biomarkers that can predict AKI after AMI may facilitate immediate interventions. Recently, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), and klotho have been established as novel AKI biomarkers. However, their effects have not been studied in patients presenting with AMI. In this study, we will measure the serum levels of these three biomarkers to find reliable biomarkers for early diagnosis of AKI in AMI patients. Methods: This prospective observational cohort study was conducted between May 2016 and November 2017. A total of 285 consecutive patients with AMI were enrolled. The study was approved by the institutional review board of Peking University People’s Hospital (No. 2016PHB 042-01). AKI was defined according to the KDIGO criteria in 2012. At admission, the clinical data of patients was collected and serum levels of several AKI biomarkers, including cystatin C, NGAL, and klotho, were measured by ELISA. The relationship between biomarker levels of AKI were analyzed and their discrimination performances were compared. Results: AKI incidence was 17.5% (50/285) during hospitalization. Compared to patients without AKI, the AKI group had higher mortality (20.0% vs. 0.4%, p < 0.001) and tended to be older, had higher incidence of chronic kidney disease, severe cardiac function, more cardiac complications, larger doses of diuretics, and less use of angiotensin-converting enzyme inhibitors/angiotensin receptor blocker and statins. Moreover, AKI patients experienced an increase in serum cystatin C (3,709.2 ± 2,281.5 vs. 1,918.5 ± 1,140.6 ng/mL, p < 0.001), NGAL (118.0 ± 70.3 vs. 91.8 ± 52.3 ng/mL, p = 0.003), and klotho (742.2 ± 497.4 vs. 470.3 ± 257.2 pg/mL, p <0.001). Furthermore, the areas under the receiver operating curves demonstrated that serum cystatin C levels at admission had modest discriminative powers for predicting AKI after AMI compared with serum creatinine (0.899, 95% CI, 0.855–0.944 vs. 0.734, 95% CI, 0.649–0.819, p <0.001). There was no difference between the discrimination performances of serum creatinine, NGAL, and klotho. Conclusion: Elevated cystatin C levels are associated with AKI in patients with AMI. This study provides reliable evidence that cystatin C levels may be superior to serum creatinine for predicting AKI after AMI at admission.