A time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin B1 and an application thereof are disclosed. The kit comprises a time-resolved fluorescent immunochromatographic test strip and sample reaction vials each containing a europium-labeled monoclonal antibody lyophilized product; wherein the fluorescent test strip comprises a PVC substrate, and a surface of the PVC substrate is adhered with a water absorbing pad (1), a detection pad (2) and a sample pad (3) from top to bottom, adjacent pads being connected and overlapping at connections. The detection pad (2) adopts a nitrocellulose membrane as the base thereof and is arranged with a lateral quality control line (5) and five detection lines (5, 6, 7, 8, 9) from top to bottom each covered by a bovine serum albumin conjugate of each toxin. The fumonisin B1 monoclonal antibody is generated by the hybridoma cell strain Fm7A11 having China Center for Type Culture Collection (CCTCC) accession number C201636. The kit is applicable to simultaneous detection of mixed contamination of aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone and sterigmatocystin.