The invention discloses a time-resolved fluorescence immunochromatography reagent used for rapid quantitative detection of CK-MB, and a preparation method thereof, and belongs to the field of clinical diagnose. The time-resolved fluorescence immunochromatography reagent is composed of a test paper strip and a fluorescent liquid; the test paper strip comprises a base plate, Fusion5, a nitrocellulose membrane, and a water absorption pad; Fusion5, the nitrocellulose membrane, and the water absorption pad are connected in sequence in the horizontal direction, and are fixedly arranged on the base plate; the nitrocellulose membrane is coated with a CK-MB monoclonal antibody 1 detection line and a quality control line composed of rabbit IgG antibodies; and the fluorescent liquid comprises CK-MB monoclonal antibody 2 labelled fluorescent microspheres, and goat anti-rabbit antibody labeled fluorescent microspheres. According to the time-resolved fluorescence immunochromatography reagent, time-resolved fluorescent microspheres are used for increasing fluorescence intensity, reducing background signals, and realizing quantitative detection of CK-MB content of whole blood, serum, or blood plasma at the same time; and only 10 to 20ml of a sample is needed. Application of the time-resolved fluorescence immunochromatography reagent is convenient and rapid; operation is simple; detection time is short; specificity and sensitivity are high; detection results are more accurate; and the time-resolved fluorescence immunochromatography reagent is suitable for clinical POCT rapid diagnosis.