Three-dimensional (3D) cultured tumor spheroid models, as one type of in vitro model, have been proven to have more physiological similarities to in vivo animal models than cells in 2D cultures. Tumor spheroids have been widely used in preclinical experiments of anticancer drug treatments, providing reliable data in pathogenetic research. Currently, different 3D cell culture conditions, even in the same cell line, generate heterogeneous spheroids in morphology and size, resulting in different growth rates or drug-killing responses. Therefore, the measurement and evaluation of the properties of tumor spheroids have become highly demanding tasks with huge challenges. For functional characterization of tumor spheroids, the microenvironment sensitivity and quantitative properties of the fluorescence lifetime microscopy imaging (FLIM) technique have great advantages for improving the reliability of cell physiological testing. In this paper, we have proposed a FLIM-based approach to observe the lipid components labeled with Nile red of cells in both 3D and 2D cultures. The imaging data and analysis provided basic information on the sizes, morphologies, and cell membrane fluorescence lifetime values of the tumor spheroids. FLIM data showed that the microenvironment of the cell membrane in the 3D model was largely altered compared to that in the 2D culture. Next, a series of parameters that may influence the lipid components of tumor cells and tumor spheroids were tested by FLIM, including pH, viscosity, and polarity. The results showed that pH and viscosity contributed little to the change in fluorescence lifetime values, while the change in cell membrane polarity was the main cause of the alterations in fluorescence lifetime data, suggesting that cell membrane polarity should be considered a marker in distinguishing tumor spheroids from cellular physiological status. In conclusion, this FLIM-based testing process has been proven to be a quantitative method for measuring the differences between the cells of the 3D model from the 2D cultured cells with satisfactory sensitivity and accuracy, providing a high potential standard assay in the quality evaluation and control of tumor spheroids for future anticancer drug development.