Chronic kidney disease (CKD) represents a significant global health burden. Currently employed CKD biomarkers are influenced by various factors and lack accuracy in reflecting early-stage renal fibrosis severity. Consequently, there is an urgent need for the identification of early, noninvasive CKD biomarkers. Urine, easily collectible and kidney-derived, has demonstrated potential as a diagnostic source for various kidney diseases by leveraging its RNA content. To address this, we obtained RNA-seq data pertaining to urinary RNAs from both CKD patients and healthy controls via the Gene Expression Omnibus database (GEO). The DEseq2 software was utilized to identify differentially expressed RNAs (DE-RNAs). To evaluate the overall accuracy of these DE-RNAs in urine, we performed Receiver Operating Characteristic analysis (ROC). Selected urinary RNAs were subsequently validated using reverse-transcription quantitative real-time Polymerase Chain Reaction (qRT-PCR) in conjunction with ROC analysis. Computational and experimental analyses revealed significant increases in miR-542-5p, miR-33b-5p, miR-190a-3p, miR-507, and CSAG4 within the urine of CKD patients, exhibiting high AUC values. In conclusion, our findings suggest that urinary RNAs hold promise as diagnostic biomarkers for CKD.© 2023. The Author(s).