The invention discloses a diagnosis kit for breast cancer and preparation method thereof. Five kinds of tumor markers for diagnosis of breast cancer such as CA15-3, CEA, CA19-9, CA12-5, SF are detected together at the same test paper and using immunity chromatography technique the monoclonal antibodies for CA15-3, CEA, CA19-9, CA12-5, SF are fixed on the cellulose nitrate film, at the same time the five kinds of monoclonal antibodies are marked by gold particles of different size and the said gold antibodies are mixed to produce gold affixture pad, finally the gold affixture pad is assembled with a sample treating pad to form a test paper. The said kit quickly test the five kinds of antigens in human whole blood or serum, plasm using double antibody sandwiching method, so as to conquer many defects of previously separation detecting method, only one drop of blood can detect five tumor markers and the detection result is more direct and the sensitivity and specificity of detection is greatly increased.