Urothelial carcinoma is a common cancer of the urinary system, and its incidence in Taiwan has been increasing. A major challenge in cancer treatment is the variability in patient responses to the same chemotherapy drugs, which limits the effectiveness of standardized therapies. Conventional drug testing methods, such as two-dimensional cell cultures and animal models, fail to accurately represent the human tumor microenvironment, leading to poor clinical translation. In this study, patient-derived tumor organoids were established to evaluate individualized drug responses and support precision medicine. Tumor tissues from three urothelial carcinoma patients were used to generate three-dimensional organoids that preserve tumor structure, heterogeneity, and molecular characteristics. Organoid morphology and pathological features were analyzed and compared with the original tumors. Four commonly used chemotherapy drugs were tested to assess inter-patient differences in drug sensitivity. In addition, organoids were cultured in a microfluidic chip system to simulate dynamic blood flow conditions. The established organoids exhibited distinct growth patterns and morphological characteristics, reflecting individual tumor differences. Pathological staining confirmed strong similarity between organoids and original tumor tissues. Drug response assays revealed significant variability in chemotherapy sensitivity among patients. Organoids cultured in the microfluidic chip system remained stable and viable under dynamic conditions. These results demonstrate that patient-derived tumor organoids, particularly when integrated with microfluidic technology, provide a physiologically relevant platform for personalized drug testing and have strong potential to advance precision cancer therapy.