Bladder cancer (BCa) is the second most common urological malignancy, but the techniques used today to detect and monitor BCa are frequently invasive and/or have inadequate sensitivity and specificity. Therefore, it is imperative to create a noninvasive test that is both sensitive and accurate for diagnosing BCa. This research introduces and validates the diagnostic performance of H4C6/SOX1-OT gene methylation in the diagnosis of BCa based on urine samples by designing two parts of studies: the case-control study and the prospective validation study. In the case-control study, the methylation test of H4C6/SOX1-OT achieved a sensitivity/specificity/positive predictive value (PPV)/negative predictive value (NPV) of 87.9%(95%CI, 79.4%-93.3%)/90.4%(95%CI, 80.7%-95.7%)/92.6%(95%CI, 84.8%-96.7%)/ 84.6%(95%CI, 74.3%-91.5%) (kappa value 77.6%). The sensitivities for low grade, high grade, Ta-T1, and T2-T4 were 85% (17/20), 88.6% (70/79), 85.4% (41/48) and 92.6% (25/27). Statistical analysis showed the diagnostic sensitivity of test was not affected by sex, age, tumor grade or tumor stage (P >
0.05). In the prospective validation study, the H4C6/SOX1-OT methylation test yielded an overall sensitivity/specificity/PPV/NPV of 84.8%(95%CI, 67.3%-94.3%)/90.0%(95%CI, 75.4%-96.7%)/87.5% (95%CI, 70.1%-95.9%)/84.6% (95%CI, 73.0%-95.4%) (kappa value 75.0%), indicating 38.4% of spared cystoscopy. These findings highlight the potential of the H4C6/SOX1-OT methylation in urine DNA as a promising molecular diagnostic tool for detecting BCa, especially for early-stage tumors, which may reduce the need for cystoscopy.