Objective Optimizing the preparation method of mouse lung cryosections to improve the quality of lung cryosections helps to enhance the specificity of immunofluorescence staining and obtain more accurate and reliable experimental results. Methods C57BL/6 mice were used to make cryosections by the traditional post-freezing fixation method, the pre-freezing fixation method, and a modified perfusion pre-freezing fixation method, respectively. A laser scanning confocal fluorescence microscope was used to observe lung tissue immunofluorescence staining. The whole areas of mouse lung slices were scanned by fluorescence microscope, and then the numbers of intact airways per unit area of lung tissue were calculated. Results For the lung cryosections made by the traditional post-freezing fixation method, the alveoli structure was damaged, and the airway wall was seriously broken with non-specific staining. The lung cryosections made by the pre-freezing fixation method showed relatively intact alveolar and airway structures but collapsed alveoli and several destroyed airways. The structure and morphology of the alveoli and airways were intact and clear in the lung cryosections prepared by the modified perfusion pre-freezing fixation method. In addition, the locations of target genes were accurate with multiple immunofluorescence staining. The number of intact airways (diameter ≥100 μm) per unit area in the lung cryosections from the modified perfusion pre-freezing fixation method was higher than from 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 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 is beneficial to maintain mouse lung tissue's morphological integrity and obtain high-quality multiplex immunofluorescence staining results.