Background <p>While urine fluorescence in situ hybridization (FISH) has established itself as a crucial diagnostic modality for upper tract urothelial carcinoma (UTUC), the broader clinical applications of this technique are yet to be fully elucidated.</p> Methods <p>This retrospective cohort study analyzed patients who underwent urine FISH test at Qilu Hospital of Shandong University from 2022 to 2025. We assessed its clinical value by constructing ROC curves. Additionally, a novel predictive tool based on urine FISH features and blood routine examination indicators was then developed to enhance urine FISH’s staging predictive ability. We also explored the correlation of urine FISH features with LVI, Ki67, and HER2.</p> Results <p>The study encompassed 220 patients, revealing the utility of urine FISH test in informing clinical treatment. Particularly, among the urine FISH features evaluated, CEP7 demonstrated superior performance in identifying patients requiring radical nephroureterectomy (AUC = 0.885), distinguishing renal pelvis carcinoma (AUC = 0.926), and discriminating non-UTUC individuals (AUC = 0.911). Regrettably, urine FISH features demonstrated constrained predictive efficacy in UTUC staging. Consequently, leveraging machine learning, we developed a staging prediction tool based on urine FISH features and blood routine examination indicators, with the SVM model demonstrating optimal predictive performance. In addition, we observed that certain urine FISH features were associated with the expression levels of HER2 and Ki67 in neoplastic tissues.</p> Conclusions <p>Our study provides an initial exploration into the extended utility of urine FISH beyond conventional diagnostics in UTUC, thereby offering novel insights for the advancement of precision oncology in this disease.</p> Clinical trial number <p>Not applicable.</p> Retrospectively registered <p>KYLL-202412-044.</p>