Alternations of lymphocyte in biophysical properties (e.g., morphology and viscoelasticity) are related to the human health, disease diagnosis and treatment. Here, we used atomic force microscopy (AFM) to characterize the morphology and mechanical properties of normal lymphocyte and Jurkat. The AFM images revealed that their cell shapes appeared similar. The mechanical properties of the two groups were tracked with AFM-based force spectroscopy. The normal lymphocyte cells had a high adhesion force distribution in (796.7±248.5) pN, whereas the Jurkat cells had a low force distribution in (158.5±37.5) pN. The adhesion force revealed that the Young’s modulus of normal lymphocyte cells (0.471±0.081 kPa) was nearly four times higher than that of Jurkat cells (0.0964±0.0229 kPa) at the same loading rate. The stiffness of normal lymphocyte cells was (2.278±0.488) mN/m and that of Jurkat cells was (4.322±0.382) mN/m. The differences in mechanical properties of normal and cancerous cells were obvious that healthy and diseased states could be clearly distinguished. These results may be applied to the clinic disease diagnosis for distinguishing the normal cells from the cancer ones even when they show similar shapes.