The invention discloses a preparation kit and preparation method of a cell group mainly comprising CD4<+>T cells and a Th1 cell subset, a use of the Th1 cell subset in preparation of an anti-tumor cell preparation, and belongs to the field of biotechnology. The preparation method utilizes a CD4 monoclonal antibody and a culture bottle made of polystyrene, polyvinyl chloride or polyethylene to realize preparation of the cell group mainly comprising CD4<+>T cells, and utilizes two cytokine combination to realize preparation of the Th1 cell subset. Through use of the kit and preparation method provided by the invention, high-ratio CD4<+>T cells in a single karyocyte are obtained. The obtained Th1 cell subset has strong immunocompetence, can secrete a large amount of cell granzyme B, perforin, IFN-gamma and TNF-alpha and has substantial anti-tumor activity.