The invention relates to an anti-CD20 monoclonal antibody, a preparation method and an application of the anti-CD20 monoclonal antibody; Chinese hamster ovary cells CHODG44 are adopted to prepare the anti-CD20 monoclonal antibody consistent to rituximab, thereby establishing a large-scale high-expression production technology for 300L of eukaryocytes, wherein the protein expression quantity is more than 1.2g / L and the protein purification yield is improved to more than 60%. The anti-CD20 monoclonal antibody is safe, effective, relatively strong in practicability, low in cost, convenient and quick, and the preparation technology of the anti-CD20 monoclonal antibody is simple and convenient, easy to operate and use and obvious in curative effect, thereby providing a new source of medicines for the B cell induced malignant tumors, particularly the non-hodgkin lymphoma, autoimmune disease, renal failure, hepatitis B, hepatitis C and diabetes, etc.; the anti-CD20 monoclonal antibody is applied to the large-scale production and commercial application in the industries such as medicine, biotechnology and the like and has a good application prospect, remarkable social benefit and economic benefit.