Lay Description In the targeted therapy of B-cell non-Hodgkin lymphoma(NHL), rituximab is a powerful antibody drug which was approved by USFDA in 1997. The response rates and survival outcomes of B-cell NHL patients were remarkably improved after the clinical use of rituximab, either by itself or when combined with chemotherapy. Rituximab is now the mainstream therapy strategy for B-cell NHLs. But there are still many patients who have no responses or develop resistance to rituximab and developing new antibodies with enhanced efficacies to provide effective therapies for those rituximab-insensitive patients has become an important issue which is urgently needed to be solved. Consequently, investigating the underlying mechanisms of rituximab's killing effect is of great significance. Traditional biochemical experiments were performed on cells grown in vitro. While we know the environment of cells grown in vitro was quite different from the environment of cells in vivo, and thus the experimental results obtained from cells grown in vitro may not faithfully reflect the cellular and molecular behaviors in vivo. Viewed from this aspect, investigating the behavior directly on clinical patient cancer cells will help us to better understand the mechanisms of drug action. In this work, we used atomic force microscopy (AFM) to probe the drug-target interactions directly on cancer cells prepared from clinical B-cell NHL patients and the experimental results indicated that the distribution of target on the cancer cells was related to the clinical efficacies of rituximab, providing novel insights in understanding the rituximab's biological functions. Summary Rituximab is an exciting monoclonal antibody drug approved for treating B-cell lymphomas and its target is the CD20 antigen which is expressed on the surface of B cells. In recent years, the variable efficacies of rituximab among different lymphoma patients have become an important clinical issue and urgently need to be solved for further development of antibodies with enhanced efficacies. In this work, atomic force microscopy (AFM) was used to investigate the nanoscale distribution of CD20 on the surface of tumour B cells from lymphoma patients to examine its potential role in the clinical therapeutic effects of rituximab. By performing ROR1 fluorescence labelling (ROR1 is a specific tumour cell surface marker) on the bone marrow cells prepared from B-cell lymphoma patients, the tumour B cells were recognized, and then AFM tips carrying rituximabs via polyethylene glycol crosslinkers were moved to the tumour cells to probe the specific CD20-rituximab interactions. By applying AFM single-molecule force spectroscopy (SMFS) at the local areas (500x500 nm(2)) on the surface of tumour B cells, the nanoscale distributions of CD20 on the surface of tumour B cells were mapped, visually showing that CD20 distributed heterogeneously on the cell surface. Bone marrow cell samples from three clinical B-cell lymphoma cases were collected to analyze the binding affinity and nanoscale distribution of CD20 on tumour cells. The experimental results showed that CD20 distribution on tumour cells were to some extent related to the clinical therapeutic outcomes while the CD20-rituximab binding forces did not have distinct effects to the clinical outcomes. These results can provide novel insights in understanding the rituximab's clinical efficacies from the nanoscale distribution of CD20 on the tumour cells at single-cell and single-molecule levels.