The invention relates to the technical field of biochemical detection, in particular to a detection kit for aspartate aminotransferase with good repeatability and a preparation and application methodthereof. The kit comprises a reagent R1 and a reagent R2. The reagent R1 comprises a buffer solution, a PH value adjusting agent, L-aspartic acid, potassium ferrocyanide, an AP5A, an EGTA, lithium chloride, a surfactant, levoglucosides, lactate dehydrogenase, malate dehydrogenase, and a preservative component. The reagent R2 comprises a buffer solution, a PH value adjusting agent, a BSA, a preservative, a-ketoglutarate, and NADH components. The kit is optimized in terms of removing reflection interference, increasing the activity of the reacted enzyme, improving the uniformity of the reactionsystem, and preferably selecting the excellent sub-wavelength. The kit remarkably improves the repeatability of reagents, especially the repeatability of low-value samples so as to improve the accuracy of clinical diagnosis results and achieve great clinical practical value.