Abstract: Objective To evaluate the application of the HB&L microbial rapid cultivation system combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the diagnosis of urinary tract infection (UTI) and compare it with the traditional urine culture method, clarifying its advantages in diagnostic efficiency, accuracy, and clinical application. Methods A total of 202 urine samples from suspected UTI patients were collected from April to May 2024 at the Affiliated Hospital of Xuzhou Medical University. After excluding unqualified samples, 177 cases were included. Traditional urine culture was used as the gold standard, and the diagnostic efficiency of the HB&L system was analyzed. Results Among the 177 samples, the positive detection rate for traditional urine culture was 26.55% (47/177), while the positive detection rate for the HB&L system was 27.12% (48/177), with no statistically significant difference (P>0.05). The sensitivity of the HB&L system was 97.87%, specificity was 98.46%, positive predictive value was 95.83%, negative predictive value was 98.46%, and the overall consistency with traditional methods was 98.31% (Kappa=0.89). Half of the positive samples were reported within 2 h. The HB&L system achieved 100% detection rate for gram-negative bacilli and fungi; only one case of Gardnerella vaginalis resulted in a false negative. Conclusions The HB&L system combined with MALDI-TOF MS provides a rapid and accurate method for UTI diagnosis, significantly reducing diagnostic time. The consistency in detecting and identifying single bacterial infections is high, making it a promising new method for rapid UTI diagnosis in clinical settings.