Background/Aim. Prompt and precise detection of urinary tract infections (UTIs) is crucial for timely antibiotic administration and improved treatment outcomes. The aim of the study was to analyze the diagnostic efficacy of fluorescence rapid on-site evaluation (ROSE) compared to bacterial culture (BC) and to evaluate its clinical applicability for identifying UTIs. Methods. This retrospective study included 240 inpatients with clinically indicated UTIs evaluated between October 2024 and March 2025. All urine specimens were analyzed using both fluorescence ROSE testing and BC, with the latter serving as the reference standard. Diagnostic metrics–such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy–were calculated. Receiver operating characteristic (ROC) curve analysis was performed to determine the area under the curve (AUC). Results. BC revealed 179 (74.58%) positive cases. Fluorescence ROSE demonstrated a sensitivity of 95.53%, specificity of 85.25%, PPV of 95.00%, and NPV of 86.67%, resulting in an overall diagnostic accuracy of 92.92%. The ROC analysis produced an AUC of 0.90 (95% confidence interval: 0.86–0.94). The misclassification rate was minimal at 7.08%. Escherichia coli was the primary pathogen, constituting 54.19% of isolates. Furthermore, fluorescent ROSE significantly decreased diagnostic turnaround time compared to traditional culture. Conclusion. Fluorescence ROSE has improved diagnostic precision and fast detection capabilities for UTIs. As an adjunct to BC, it may improve early identification and facilitate rapid antibiotic therapy, especially in clinical settings requiring a swift response.