Objective To observe the effect of different fixative solutions on cancer cell morphology and membrane permeability. Methods Human pancreatic acinar epithelial carcinona(HPAC) cells of human pancreatic cancer and HeLa cells of human cervical cancer were fixed with 4 fixation solutions: freshly prepared 0.25% paraformaldehyde solution; Freshly prepared 4% paraformaldehyde solution; 75% ethanol solution; 90% ethanol solution. The fixation time is 30 minutes. PBS solution and complete medium were used as the controls. Cell morphology of each group was observed under optical microscope. Changes in cell membrane permeability were observed by fluorescence staining with 7-aminoactinomycin (7-AAD), which is not cell membrane permeable in intact cells but permeable in damaged cells. Hoechst33342 was used for staining both intact and damaged cells. Results The cells in the complete medium group were similar to unfixed cells in morphology, and the fluorescence staining of 7-AAD was the weakest. The cells in the complete medium group have typical cell morphology and low 7-AAD permeability. The 0.25% paraformaldehyde solution group had similar cell morphology to the complete medium group, and the 7-AAD fluorescence staining was weak. The morphology of cells in the 4% paraformaldehyde solution group was typical, but the fluorescence staining of 7-AAD was strong. The cells in the 90% ethanol solution group showed swelling, with a larger volume than the unfixed cells and a stronger fluorescence staining of 7-AAD. The cell swelling in 75% ethanol solution group was not as obvious as that in 90% ethanol solution group, and the fluorescence staining of 7-AAD was strong. The cells in PBS group were round, and the fluorescence staining of 7-AAD was strong. Conclusion 0.25% paraformaldehyde solution can not only fix tumor cells, but also maintain the integrity of cell membrane.