The invention discloses a time-resolved fluorescence immunochromatography test strip for detecting acetamiprid, and a preparation method and an application thereof. The test strip comprises a bottom plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption, which are sequentially lapped and adhered on the bottom plate, wherein an acetamiprid monoclonal antibody labelled by a fluorescent microsphere is buried on the conjugate release pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, the detection area is coated with acetamiprid hapten-carrier protein conjugate, the quality control area is coated with a goat anti-mouse anti-antibody, and the acetamiprid hapten is obtained by a reaction of N-noracetamidine and an aminobutyric acid. The invention further provides a preparation method of the test strip and a method for detecting the acetamiprid in a sample by using the above test strip. The test strip and the detection method provided by the invention have the advantages of simple operation, high sensitivity, high detection speed and low cost, and can be used for realizing rapid detection and onsite monitoring of the acetamiprid in mass samples.