Objective To explore the comparative effect of automatic fluorescence staining method and traditional wet mount microscopic examination in vaginal secretion test. Methods 6 672 patients who received vaginal secretion examination in the hospital from November 2023 to November 2024 were selected as the samples for study. All samples were examined by automatic fluorescent staining and traditional wet microscopy, with clinical diagnosis as the gold standard. The detection conditions of different vaginal microbial infection and vaginitis were compared between the two detection methods, and the diagnostic value of the two methods on vaginitis was analyzed. Results The detection rates of clue cells, Candida and trichomonas by automatic fluorescence staining were significantly higher than those by traditional wet microscope method. The total detection rate of bacterial vaginosis, vulvovaginal candida and trichomoniasis vaginitis by automatic fluorescent staining method was significantly higher than that of traditional wet tablet method. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value and Kappa value in the diagnosis of vaginitis were 96.78%, 99.89% 98.97%, 99.74%, 98.65% and0.975 of automatic fluorescence staining method, and were 81.84%, 97.44%, 92.79%, 93.13%, 92.67% and 0.821 of traditional wet mount microscopic examination. The consistency between automatic fluorescence staining method and clinical diagnosis was high. Conclusion Compared with traditional wet mount microscopic examination, automatic fluorescence staining method has higher detection rates of vaginal microorganisms and higher diagnostic value on vaginitis, thus the latter one is more suitable for clinical diagnosis.