Objective To explore the application of RT-F2000 (automatic vaginal secretions detector) in the detection and diagnosis of pathogenic microorganisms in gynecological urogenital tract. Methods The clinical data of 120 patients who underwent gynecological examination in Tianzhu County People’s Hospital were retrospectively analyzed, and the samples were selected from January 2023 to November 2023. After admission, a total of 120 samples of genital tract secretions and urethra samples were collected. RT-F2000 and microbial culture method were used to detect pathogenic microorganisms in the genitourinary tract. The detection of pathogenic microorganisms was compared between RT-F2000 and microbial culture method. Microbial culture method was used as the gold standard to evaluate the diagnostic efficacy of RT-F2000. Results (1) The results of 120 samples of secretions showed that 83 positive specimens were detected by microbial culture method, with a positive rate of 69.16%. There were 34 cases (40.96%) of mycoplasma, 25 cases (30.12%) of mold, 14 cases (16.87%) of cue cells and 10 cases (12.05%) of trichomonas, respectively. The positive rate of RT-F2000 was 70.83% (85 cases). There were 35 cases (41.17%) of mycoplasma, 25 cases (29.41%) of mold, 15 cases (17.64%) of cue cells and 10 cases (11.76%) of trichomonas, respectively. There was no statistical significance in the positive rate between the two methods (P < 0.05). (2) Of the 83 positive specimens by microbial culture method, 70 were infected with simple pathogens and 13 were infected with mixed pathogens, respectively, with a composition ratio of 84.34% and 15.66%. In the 85 positive samples by RT-F2000, 72 cases were infected with simple pathogens and 13 cases were infected with mixed pathogens, respectively, with a composition ratio of 84.71% and 15.29%. (3) Compared with the microbial culture method, the coincidence rate of RT-F2000 detection of mycoplasma was 99.17%; The coincidence rate of mold was 100.00%. The cue cell coincidence rate was 99.17%. The coincidence rate of trichomonas was 100.00%. (4) The Kappa value of mycoplasma of RT-F2000 detection was 0.961. The Kappa value of mold was 0.938. The Kappa value of the cue cells was 0.952. The Kappa value of trichomonas was 0.974, which was in good agreement with that of microbial culture method (P > 0.05). Conclusion The application of RT-F2000 in the detection of pathogenic microorganisms in the gynecological urogenital tract can realize rapid and accurate diagnosis of pathogens in the patient’s reproductive tract, which reflects high clinical application value.