The present invention discloses a humanized anti-CD20 antibody, which comprises a heavy chain comprising a polypeptide according to one of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 36; and a light chain comprising apolypeptide according to one of SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 37. Comparing to murine-derived antibodies and human-mouse chimeric antibodies, said humanized anti-CD20 antibody maintains or improves high binding activity of the variable regions, meanwhile reduces the immunogenicity of chimeric antibodies, consequently achieves the effect of reducing medicine side effects and improving clinical treatment. The antibody disclosed by the present invention is efficiently expressed in animal cells, can be used for industrial production. It could be used in treating B cell lymphoma, leukaemia, or B cell-associated autoimmune disease with a wide application prospect.