Background: According to European Association of Urology (EAU) guidelines, repeated transurethral resection of bladder tumor (re-TURBT) is recommended in a large percentage of high-risk non–muscle-invasive bladder cancers (NMIBC) due to possibility of incomplete initial TURBT. But so far, no diagnostic tests able to improve patients’ stratification exist to reduce negative biopsy ratio of re-TURBT. To evaluate the value of the OncoUrine test for the predictive capacity of biopsy findings on re-TURBT, a prospective multi-center clinical trial was designed. Methods: This was a blinded, observational, prospective multicenter study (NCT05112523). Between December 2020 and September 2022, patients with a confirmed diagnosis of high-risk NMIBC at three study centers were enrolled. The inclusion criteria were ≥age 22 years old, prepare for re-TURBT following EAU guidelines, able to produce 100 ml of morning urine, and able to consent. Urine samples were collected from the enrolled patients for the OncoUrine tests before re-TURBT, and the test results were compared with the pathological results to calculate the performance. High-throughput sequencing of 17 genes and methylation analysis for ONECUT2 CpG sites were combined as a liquid biopsy test panel named OncoUrine. Results: Out of 109 patients enrolled, 93 were eligible for the performance analysis. In 33 cases, residual tumors were confirmed while the remaining 60 patients was found to have a result of negative pathological section. In the overall OncoUrine test results, variants from 11 genes showed positive mutations. TERT C228T, TP53, TERT C250T, ERBB2 and PIK3CA were on the top 5 of the list with other mutations from FGFR3, KRAS, HRAS, ERBB3,ERCC2, U2AF1, and ROHA. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the OncoUrine test for predicting pathological diagnosis were 81.8% (27/33), 81.7% (49/60), 89.1% (49/55), and 71.1% (27/38), respectively. Conclusions: Even for high-risk patients for whom re-TURBT is currently recommended, the OncoUrine test can accurately differentiate between cases with incomplete primary surgeries and those with no residual tumors. An interventional clinical trial is needed to further confirm the clinical value of this urine-based biopsy method on improving the selections of NMIBC patients. Clinical trial information: NCT05112523.