Objective Optimizing the preparation method to improve the quality of mouse lung cryosections to help enhance the specificity of immunofluorescence staining and obtain more accurate and reliable experimental result. Methods C57BL/6 mouse lung tissue was used to make cryosections via the traditional post-freezing fixation method, prefreezing fixation method, and a modified perfusion pre-freezing fixation method. A laser-scanning confocal microscope was used to observe lung tissue immunofluorescence staining. Whole areas of mouse lung slices were scanned by fluorescence microscope, and the numbers of intact airways per unit area of lung tissue were calculated. Results In the lung cryosections made via the traditional post-freezing fixation method, the alveoli structure was damaged, the airway wall was seriously disrupted, and there was non-specific staining. Lung cryosections made via the pre-freezing fixation method showed relatively intact alveolar and airway structures but collapsed alveoli and several destroyed airways. In the lung cryosections obtained via the modified perfusion pre-freezing fixation method, the structure and morphology of the alveoli and airways were intact and clear. Additionally, the locations of multiple proteins targeted with immunofluorescence staining were accurate. The number of intact airways (diameter ≥100 μm) per unit area in the lung cryosections obtained via the modified perfusion pre-freezing fixation method was higher than that from cryosections made using the pre-freezing fixation method ((0. 66±0. 15) / mm2 vs (0. 33±0. 14) / mm2, P< 0. 05) and was also significantly higher than that from sections made using the traditional post-freezing fixation method ((0. 66 ± 0. 15) / mm2 vs (0. 02 ± 0. 04) / mm2, P< 0. 01). Conclusions The modified perfusion pre-freezing fixation method for cryosections is conducive to maintaining the integrity of mouse lung tissue morphology and obtaining high-quality multiplex immunofluorescence staining result.