The invention discloses a preparation method of biological agent for inhibiting WT1 gene mutation. The preparation method comprises the following steps: separating T lymphocytes from blood of the human body; carrying out multiplication culture on the T lymphocytes in a culture solution; carrying out domestication on the cultured lymphocytes and a cell strain in a culture bottle; testing the T lymphocytes domesticated by the cell strain and cancer cells together, and after the T lymphocytes have ability of attacking the cancer cells, separating the T lymphocytes from the cancer cells; carryingout mixing testing on the separated T lymphocytes on WT1 gene, wherein the domesticated T lymphocytes have ability of resisting WT1 gene mutation; and separating the T lymphocytes with resisting and attaching ability from the culture solution, preparing an active preparation from the T lymphocytes and human immunoglobulin together, and refrigerating the active preparation in a sealed manner. The preparation method of the biological agent for inhibiting WT1 gene mutation has the advantages that WT1 gene mutation is inhibited from the source, cancel cells are stopped, cancers can be prevented effectively, the prevention effect can reach 95% or above, and the result is accurate.