The invention provides a nucleic acid aptamer fluorescence test strip, a method for preparing the same and application of the nucleic acid aptamer fluorescence test strip, and relates to the technicalfield of biological detection. The nucleic acid aptamer fluorescence test strip comprises a bottom plate, a sample pad, a nanometer material pad, a reaction film and a water absorption pad. The reaction film is provided with a detection line, and the detection line is arranged at the end, which is close to the nanometer material pad, of the reaction film; nucleic acid aptamer with fluorescein labels and/or nucleic acid aptamer with fluorescence labels and linker sequences and free fluorescein are attached to the nanometer material pad, and complementary strains of nucleic acid aptamer or complementary strains of linker sequences of nucleic acid aptamer are fixed to the detection line. The method for preparing the nucleic acid aptamer fluorescence test strip includes sequentially adheringthe sample pad, the nanometer material pad, the reaction film and the water absorption pad on the same side of the bottom plate. The nucleic acid aptamer fluorescence test strip, the method and the application have the advantages that the nucleic acid aptamer fluorescence test strip is simple in structure, sensitive in detection and high in accuracy; the method is easy to implement and high in flexibility.