The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20⁺B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20⁺B lymphocytes was much higher than that of B-CLL CD20⁺B cells. The AFM data show that ultrastructure of B-CLL CD20⁺B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20⁺B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20⁺B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.