Background: Quinazolines, which process a wide spectrum of biological properties such as antibacterial, antifungal, antivirus, and anticancer activities, are considered one of the most important heterocycles in medicinal chemistry. Here, we described for the first time the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazoline-4-one) which emits green fluorescent in the cytosol and exhibits phototoxicity toward human bladder cancer (BC) cells under blue-light exposure. Materials and Methods: Human BC cells (5637 and T24) and immortalized uroepithelial cell (SV-HUC1) were utilized in this study. To trace the localization of MJ-56, MitoTracker and LysoTracker were applied. The cell viability with or without blue light exposure were monitored by WST-1 reagent, direct recording, and clonogenic assays. The apoptosis induction in MJ-56 treated cells was detected. Results: MJ-56 emits green fluorescent in the cytosol. Vital staining of mitochondria or lysosomes demonstrated that the MJ-56 fluorescent was not located in either organelles. MJ-56 treatment for 24 h did not cause significant loss of cell viability in BC cells. However, treatment of 0.125 μM MJ-56 for 1 h and exposed to blue light for 15 mins significantly reduced cell viability. Interestingly, our results showed that MJ-56 has minimal impact on SV-HUC1 even with the blue-light exposure. The caspase 3/7 activities in BC cells treated with MJ-56 and exposed to blue light were significantly increased 1 h post-treatment. However, the DNA fragmentation cannot be detected at 1, 6, or 24 h posttreatment due to the loss of viable cells. Conclusions: MJ-56 exhibits phototoxicity toward BC cells with minimal impact on uroepithelial cells, indicating a novel therapeutic agent against BC. The mechanism underlying MJ-56-induced cell death as well as the translational studies warrants further investigation.