The invention relates to the field of clinical diagnosis, in particular to a time-resolved fluorescence immunochromatographic reagent for rapid quantitative simultaneous detection of cTnI, CKMB and Myo, and a preparation method thereof. The reagent comprises a test strip and fluorescent liquid, wherein the test strip comprises a bottom plate, Fusion5, a nitrocellulose membrane and an absorbent pad, and the Fusion5, the nitrocellulose membrane and the absorbent pad are sequentially connected and fixed on the bottom plate in the horizontal direction. Time-resolved fluorescent microspheres are used to improve the fluorescence intensity and decrease background signals, and cTnI, CKMB and Myo indexes in whole blood, serum or plasma can be simultaneously and quantitatively detected, and a sample needs only 10 to 20mul. The test strip has the advantages of convenience, rapidness, simple operation, short detection time, strong specificity, high sensitivity and relatively accurate test results, and is suitable for rapid diagnosis of clinical POCT.