Background: Cystoscopy using white light is a standard procedure for diagnosing bladder cancer; however, white light can result in missed lesions that are present, but not visible, such as in cases of carcinoma in situ (CIS). In this case report, we describe observing the nuclei of urothelial carcinoma cells in situ that were not visible with cystoscopy under white light using probe-based confocal laser endomicroscopy (pCLE) with acrinol and fluorescein during transurethral resection of a bladder tumor (TURBT). Case Presentation: A 59-year-old male with a medical history of neurogenic bladder dysfunction with multiple bladder diverticula was referred to the urology department for gross hematuria. TURBT was performed with the assistance of pCLE, using acrinol as a novel dye. Standard cystoscopy under white light could not detect any bladder tumor; however, pCLE using acrinol could detect the abnormal nuclei of bladder CIS. Subsequent histopathologic analysis of the specimen confirmed a diagnosis of bladder CIS. To our knowledge, this is the first reported case of bladder CIS diagnosed with the assistance of pCLE using acrinol in a patient undergoing a TURBT. Conclusion: pCLE using acrinol as a novel dye can help observe the cancerous nuclei of bladder CIS that cannot be detected using conventional cystoscopy under white light. Therefore, pCLE using acrinol is one possible modality for performing an optical biopsy during TURBT.