The treatment of cancer draws interest from researchers worldwide. Of the different extracts from traditional Chinese medicines, Tubeimoside 1 (TBMS 1) is regarded as an effective treatment for cancer. To determine the mechanism of TBMS 1, the shape/pattern of HepG2 cells based on the microscopic imaging technology was determined to analyze experimental results; then the fluorescent spectra method was designed to investigate whether TBMS 1 affected HepG2 cells. A three-dimensional (3D) fluorescent spectra sweep was performed to determine the characteristic wave peak of HepG2 cells. A 2D fluorescent spectra method was then used to show the florescence change in HepG2 cells following treatment with TBMS 1. Finally, flow cytometry was employed to analyze the cell cycle of HepG2 cells. It was shown that TBMS 1 accelerated the death of HepG2 cells and had a strong dose- and time-dependent growth inhibitory effect on HepG2 cells, especially at the G2/M phase. These results indicate that the fluorescent spectra method is a promising substitute for flow cytometry as it is rapid and cost-effective in HepG2 cells.