Objectives: Cell membrane fluorescent dye known as DiO/DiD has been suggested for its stability and safety as an effective tool for live cell staining. This study developed a novel cell proliferation detection technology based on DiO/DiD. Materials and Methods: Labelling of tumor cells using membrane fluorescent dye DiO/DiD, and the fluorescence intensity was measured by usig fluorescence-activated cell sorting. The number of different tumor cell divisions was calculated through the standard curves by measuring the fluorescence intensity. Labeled cells was treated with different concentrations of doxorubicin to investigate the effect of chemotherapy on labeled cells. DiO-labeled tumor cell were injected into different organs and tissues to explore the relationship between the number of cell divisions and tumor growth. Results: Tumor cells can be divided into DiO-High and DiO-Low subgroups after chemotherapy and a higher proportion of CD133 was observed in the DiO-High cell subgroups compared to DiO-Low cell subgroups. Besides, the tumor cells from metastatic tumors proliferated more rapidly than those in situ. Conclusions: Application of cell membrane dye DiO/DiD in the quantitative detection of cell proliferation method is expected to be more feasible and applicable in future studies.