Cells perceive physical properties such as stiffness, elasticity, and topological structure of the microenvironment through the extracellular matrix, and utilize mechanosensitive proteins to respond to mechanical stimuli, thereby regulating behaviors such as proliferation, differentiation, and migration. Therefore, precise measurement of mechanical forces between cells and the matrix is crucial for studying mechanosensing and signal transduction. To measure the traction forces exerted by tumor cells on the matrix during migration, this study employs Traction Force Microscopy (TFM) technology. By real⁃time monitoring of the displacement of fluorescent microspheres embedded in the matrix and combining the mechanical properties of the matrix, high⁃precision traction force measurement at the subcellular scale is achieved using forward and inverse methods. The measurement results showed that tumor cells exerted traction forces of approximately 431.9 Pa in the protrusion regions and 153.9 Pa in the cell body regions during migration on the constructed matrix. This technology, with its advantages of high resolution, non⁃invasiveness, and real⁃time in situ detection, provides a new research approach for mechanical measurements at the micro⁃nano scale.