Background: For bladder cancer (BC), the 5-year survival rate for patients with localized tumors can reach as high as 92%, but only 45% for those with tumors spreading to nearby regions. Cystoscopy combined with tissue biopsy can miss 10-40% of the cancer cases due to multiple factors. Meanwhile, the method is invasive, causing physical discomfort and psychological trauma for the patients. Therefore, development of a noninvasive, sensitive, and specific diagnostic alternative is needed. By analyzing TCGA data and improving analytic technology currently available, we have developed an easy-to-use, convenient, and accurate detection approach for BC relying on a single methylation marker. Methods: We searched TCGA database and selected a total of 366 candidate markers including those that have been published for subsequent examination and analyzed 446 primer pairs and probe sets. Using quantitative methylight method, we narrowed the field down to top 5 performers for subsequent urine-based testing of 270 samples. Finally, two markers were selected for validation testing in a group of 530 urine samples, consisting of 161 bladder cancers, 244 normal controls, 80 renal carcinomas, and 45 carcinomas of renal pelvis and ureter. In addition, we detected the methylation level and mRNA expression of target gene in 8 bladder cancer cell lines and 1 normal bladder epithelial cell line by MSP, qMSP and qPCR. Results: Methylation marker DMRTA2 had a sensitivity of 85.7%, an AUC value of 0.924, and an accuracy of 90.6%. The specificity, from a control group consisting of patients with lithangiuria, prostatoplasia, and prostatitis as well as healthy individuals, is 93.9% (138/161). Notably, the methylation assay had the highest sensitivities for tumors at stages of T1 (94.4%) and T2 (96.6%) compared with T3 (80.0%), T4 (71.4%), and unknown stage (90.0%). While methylation was observed in 34/45 urine samples from patients with carcinomas of renal pelvis and ureter, a good sensitivity of 75.6%, it was detected at extremely low rate of 7.5% (6/80) in those with interfering cancers of kidney and prostate. Additionally, the assay specificity was significantly affected by the age of the cancer patients and selection of healthy controls, critical aspects to be carefully considered during the design and development phase of the methylation-specific testing. Compared with SV-HUC-1, the normal bladder epithelial cell line, DMRTA2 gene was hypermethylated in 8 bladder cancer cell lines, which is consistent with the results of MSP and qMSP. The mRNA levels of DMRTA2 were low in some bladder cancer cell lines, such as T24, J82, UM-UC-3 and RT4, however were fairly high levels in 5637, SCaBER, TCCSUP and SW780. Conclusions: Our data demonstrated that a single-target DNA methylation signature could be highly effective to detect bladder cancer via urine samples.