Early detection of acute kidney injury is difficult due to lack of known biomarkers; previous studies have tried to identify new biomarkers for detecting acute kidney injury at an early stage. MicroRNA, a 21-23 nucleotide noncoding RNA molecule, has emerged as a desirable marker in the diagnosis and prognosis of various diseases. This study aims to identify the expression profile of microRNA in ischemia-reperfusion-induced kidney injury and determine the possibility of using the candidate microRNA as biomarker for the detection of I/R-induced kidney injury. Based on the established rat model of I/R-induced kidney injury, a microarray analysis of rat urine was performed at the beginning of operation (0 h) as well as 72 h post operation. To validate the results, urine samples from 71 patients who underwent cardiac surgery were collected, after which urinalysis was conducted to determine the microRNA concentration. An alternative expression profile of microRNAs was detected in rat urine. The quantitative validation of microRNA showed that the expression of miR-30c-5p, miR-192-5p, and miR-378a-3p was elevated significantly in urine post operation, which was consistent with those of the microarray analysis and earlier than kidney injury molecule-1 (KIM-1). In patients with acute kidney injury, increased levels of miR-30c-5p and miR-192-5p were also detected 2 h post operation, and miR-30c-5p showed preferable diagnostic value compared with protein-based biomarkers. In conclusion, an aberrant expression profile of microRNA was detected in rat urine based on the established ischemia-reperfusion animal model. Both miR-30c-5p and miR-192-5p served as important potential diagnostic markers for I/R-induced kidney injury. Impact statement Firstly, one differentiating factor in our study is that the candidate miRNAs were screened in a controlled animal model rather than in patients with acute kidney injury (AKI) to ensure the purity of the cause of disease and to avoid possible effects of comorbidities on the spectrum of urine miRNA. This ensured the presence of only the relevant candidate miRNA (that associated with I/R injury); and what's more, the alterative expression of miR-192-5p and miR-30c-5p in animal model, patients with AKI, and cell model was confirmed simultaneously, which is likely to be more convincing. Secondly, the candidate miRNAs were screened sequentially at regular time points, which covered the initiation, progression, and partial repair stages, thus ensuring that no significant miRNAs were omitted in the screening process, and miR-biomarkers in 2 h post operation showed preferable diagnostic performance.