Background <p>Bladder carcinoma (BC) is a malignant tumor that originates from the epithelial cells of the urinary system. Currently, the main diagnostic methods suffer from disadvantages including being invasive, having high costs, and low sensitivity and specificity. Therefore, there is an urgent need to develop a non-invasive, high-performance method for diagnosing BC.</p> Materials and methods <p>We developed a urine DNA detection panel utilizing two methylation biomarkers for the diagnosis of BC. A total of 467 urine samples were collected from the First Affiliated Hospital of Anhui Medical University for DNA methylation analysis. The methylation levels of Vimentin and POU class 4 homeobox 2 gene (POU4F2) were analyzed in a training set of 306 urine samples (92 cases and 214 controls), and in an independent validation set of 161 urine samples (59 cases and 102 controls) using Real-Time PCR (RT-PCR).</p> Results <p>The Vimentin/POU4F2 combined methylation panel achieved an AUC of 0.935 (95% CI: 0.889–0.981), with sensitivity, specificity, and accuracy of 86.44% (95% CI: 0.772–0.957), 96.08% (95% CI: 0.923–0.998), and 92.55% (95% CI: 0.886–0.965) for diagnosing BC. Notably, for patients with stage I and low-grade BC, the sensitivity was 90.00% for both. Additionally, it demonstrated specificities of 96.30% and 95.83% for patients with other urinary diseases and malignancies in other systems.</p> Conclusions <p>Our study provides evidence that the two-gene methylation panel based on urine DNA detection demonstrates strong performance in diagnosing BC with high sensitivity and accuracy, offering a promising strategy for early screening and adjunctive diagnosis of BC.</p>