Background: According to European Association of Urology (EAU) guidelines, repeated transurethral resection of bladder tumor (re-TURBT) is recommended in a large percentage of non–muscle-invasive bladder cancers (NMIBC) due to possibility of incomplete initial TURBT. However, no reliable predictors have been developed to help select patient candidates who could avoid Re-TURBT in NMIBC. Methods: This was a blinded, observational, prospective multicenter study (NCT05112523). A total of 162 patients who underwent initial TURBT and were scheduled for Re-TURBT were enrolled in this study between June 2021 and August 2023 from four centers. Urine sample of each patient was collected prior to Re-TURBT. High-throughput sequencing of 17 genes and methylation analysis for ONECUT2 CpG sites were combined as a liquid biopsy test panel named OncoUrine. The OncoUrine test results were compared to pathological results at Re-TURBT to assess the performance in predicting residual tumor and predictive value of risk stratification. Patients who received intravesical infusion chemotherapy or BCG were followed up for recurrence analysis. Results: 151 patients were finally included for performance analysis. At Re-TURBT, 48 (31.8%) samples had residual tumor and 103 (68.2%) had no residual tumor. 1 Ta and 2 T1 patients were up staged to T2 at Re-TURBT. Overall, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of OncoUrine test were 77.1%, 77.7%, 61.7%, and 87.9%. Compared to OncoUrine, Cytology prior to Re-TURBT yielded a high specificity of 95.8% but lower sensitivity of 20% and combined with OncoUrine improved the sensitivity to 75.6%. In the overall OncoUrine test results, variants from 12 genes showed positive mutations. TERT, TP53, ERBB2, FGFR3, and PIK3CA were on the top 5 of the lists with other mutations from ERBB3, ERCC2, U2AF1, HRAS, KDM6A, KRAS, and ROHA. 132 were followed up with a median of 362 days (range 21 to 953). 19 patients were found recurred. The pathological results at Re-TURBT, OncoUrine, methylation and TERT results were risk stratification approaches for the recurrence analysis (positive vs. negative, p＜0.05). Conclusions: OncoUrine test after initial TURBT for NMIBC showed promise as to guide patient selection for Re-TURBT and risk stratification in the management of NMIBC. Clinical trial information: NCT05112523.