Atomic force microscopy (AFM) was used to locate CD20 molecules on the surface of lymphoma Raji cells. Rituximab (a monoclonal antibody against CD20) molecules were linked onto the AFM tip via a polyethylene glycol (PEG) linker. Raji cells were adsorbed onto glass slides coated with poly-L-lysine. First, the CD20 distribution in a local area of the cell surface was visualized using the AFM lift scan mode. Second, 16 × 16 force curves were obtained from the same cell area to construct the CD20-rituximab binding force map. Finally, free rituximab was added to block the CD20 molecules on the cell surface and the lift phase image and CD20-rituximab force map were obtained again. The experimental results indicated that when the lift height was greater than the length of the PEG linker, no recognition sites were observed in the lift phase image. However, as the lift height decreased to the length of the PEG linker, some recognition sites were observed in the lift phase image and these sites were generally consistent with the pixels in the force map. After blocking, both the recognition sites in the lift phase image and the gray pixels in the binding force map decreased markedly. These results can improve our understanding of the distribution of protein molecules on the cell surface and facilitate further investigations into cellular functions. Atomic force microscopy (AFM) was used to locate CD20 molecules on the surface of lymphoma Raji cells. Rituximab (a monoclonal antibody against CD20) molecules were linked onto the AFM tip via a polyethylene glycol (PEG) linker. Raji cells were adsorbed onto glass slides coated with poly-L-lysine. First, the CD20 distribution in a local area of ​​the cell surface was visualized using the AFM lift scan mode. Second, 16 × 16 force curves were obtained from the same cell area to construct the CD20- rituximab binding force map. Finally, free rituximab was added to block the CD20 molecules on the cell surface and the lift phase image and CD20-rituximab force map were obtained again. The experimental results that when the lift height was greater than the length of the PEG linker, no recognition sites were observed in the lift phase image. These, as the lift height decreased to the length of the PEG linker, some recognition sites were observed in the lift phase image and these sites after blocking, both the recognition sites in the lift phase image and the gray pixels in the binding force map decreased markedly. These results can improve our understanding of the distribution of protein molecules on the cell surface and facilitate further investigations into cellular functions.