Hydronephrosis is one of the most common diseases in urology. However, due to the difficulties in clinical trials and the lack of reliable in vitro platforms, the surgical indicators are not clear. Herein, the renal-on-chip with a force-sensitive resistor microfluidic platform was established to simulate the state of hydronephrosis. Cell counting kit-8 (CCK-8) and tight junction protein claudin-2 were detected on a renal-on-chip microfluidic platform with a force-sensitive resistor (ROC-FS). The results indicated that the ROC-FS had normal physiological functions and the cell viability on ROC-FS declined to around 40% after 48 h of hydronephrosis-simulated treatment. In addition, proteomics analysis of 15 clinical ureteropelvic junction obstruction (UPJO) samples showed that compared with normal children, a total of 50 common proteins were differentially expressed in UPJO children (P < 0.05, |log2fold change| ≥ 1). Metabolomic analysis of 39 clinical UPJO samples showed that a total of 241 metabolisms were dysregulated. Subsequent immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis using ROC-FS were performed to identify the clinical multi-omics results for screening. All results pointed out that the TGF-β-related signaling pathways and arginine-related metabolism signaling pathways were dysregulated and α-SMA, AGT, and AGA might be the potential biomarkers of hydronephrosis. In addition, correlation analysis of AGT and KLK1 with differential renal function (DRF) from clinical samples indicated good correlation coefficients (R2 0.923, 0.8742, 0.6412, and 0.8347). This demonstrates the state of hydronephrosis could be significantly correlated with the biomarkers. These findings could provide a reliable reference for determining surgical biomarkers clinically, and ROC could be further used in the analysis of other kidney diseases.