The invention belongs to the technical field of microbial fluorescent staining, and particularly relates to a genital tract secretion multi-fluorescent staining solution as well as a preparation method and application thereof. The genital tract secretion multi-fluorescent staining solution comprises fluorescein, nucleic acid dye, a bacteriostatic agent, an anti-quenching agent, a permeable agent and a buffer solution, and the pH is 4.5 to 6.0. The genital tract secretion multiple-fluorescence staining solution has short staining time on vaginal secretion samples, and is suitable for large-scale clinical work; the staining result is stable and reliable, the morphological structures of cells and various pathogenic microorganisms are clear, the contrast is distinct, and the morphology, size and distribution of various pathogenic microorganisms and the variety and number of various cells in the smear can be clearly observed under a microscope. In addition, the multi-fluorescent staining solution is good in stability and can be used for a long time, and the staining cost is greatly reduced.