The invention discloses a piece of fluorescence immunoassay chromatography test paper which is rapid, sensitive, simple and convenient to operate to test the residual quantity of cefalexin, and preparation of the test paper. The test paper comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorbing pad, which are adhered to a substrate in a mutual lap joint manner in sequence, wherein the nitrocellulose membrane is coated with a detection line and a quality control line; the binding pad is coated with a cefalexin monoclonal antibody with a fluorescent mark. The preparation method of the fluorescence immunoassay chromatography test paper for cefalexin residue comprises the following steps: synthesizing cefalexin-ovalbumin coating antigen, preparing a goat anti-mouse immune globulin antibody, coating the cefalexin-ovalbumin coating antigen and the goat anti-mouse immune globulin antibody onthe nitrocellulose membrane to serve as the detection line and the quality control line, preparing a fluorescence nano-particle mark cefalexin monoclonal antibody, then coating glass fiber to serve as the binding pad, sequentially adhering the sample pad, the binding pad, the nitrocellulose membrane and the water absorbing pad to a back plate in the lap joint manner in sequence, cutting the obtained test paper into the width of 4mm, and preserving at normal temperature.