Objective To explore the significance of methylation of the candidate biomarker transcription factor 21(TCF21) in bladder urothelial carcinoma. Methods From October 2016 to October 2017, 142 patients with suspected bladder cancer were selected. Among them, 80 were diagnosed as bladder cancer by pathological examination as the study group. A total of 62 non-bladder cancers were diagnosed by pathological examination as the control group. In addition, 40 healthy urine specimens during the same period were selected as the healthy group. Detected and compared the methylation of TCF21 in bladder cancer tissues, paracancerous tissues, and control tissues of the study group, and detected and compared the TCF21 methylation levels in urine of study group, control group, and healthy group. The relationship between TCF21 methylation level and clinicopathological features was explored, and the diagnostic efficacy of both for bladder cancer was analyzed. Results The methylation level of TCF21 in bladder cancer tissue was significantly higher than that in the adjacent tissue and control group (P<0.05). The methylation level of urinary TCF21 in the study group was significantly higher than that in the control and healthy groups (P<0.05). TCF21 methylation levels in bladder cancer tissues and urine were higher in men, ages>60 years, high TNM stages, and high grade bladder cancer patients (P<0.05). TCF21 methylation levels in bladder cancer tissues and urine hadhigher diagnostic efficacy for bladder cancer, and there was no significant difference in the diagnostic efficacy between the two groups (P>0.05). Conclusion TCF21 gene hasd high methylation level in urine of patients with bladder cancer and bladder cancer, and is associated with pathological features. Urinary bladder cancer tissues and urine have higher diagnostic efficacy for bladder cancer.