Background: Bladder cancer is a common malignant tumor in the urinary system. JCV has been classified as a Group 2B carcinogen due to its carcinogenicity. While JCV is mainly found in the central nervous, it has been detected in urinary epithelial tissue and urine. The NCCR is key to JCV regulation, but t its role remains unclear. Methods: Large T antigen (LTag) and P53 expression in bladder cancer tissues was detected. JCV infection and mutations were identified in bladder cancer patients using Virome. Probes specific to NCCR mutations of bladder cancer was designed, and a kit for in vitro diagnosis of bladder cancer was established. The effects of NCCR mutations on transcriptional activity were investigated by immunofluorescence, dual-luciferase reporter assays and mass spectrometry. Results: The expression of LTag and P53 were higher in JCV-positive bladder cancer. Virome analysis showed among the 8 samples, there were 3 samples in which a 5-base consecutive deletion in NCCR. Q-PCR results indicated 37 out of 45 (82.22%) bladder cancer patients were infected with JCV, with 26(57.78%) having mutations in NCCR. The results of immunofluorescence and dual-luciferase reporter assays indicated the mutations of NCCR weakened the transcriptional activity of bladder cancer. Through mass spectrometry, it was discovered the mutations of NCCR affected the binding of transcriptional regulatory proteins. Conclusion: In this study, an qPCR-assay for the detection of JCV and the subtype of NCCR was established. This approach provides a non-invasive, convenient, and accurate diagnostic method for early diagnosis and monitoring of bladder cancer.