The invention relates to the technical field of biological detection, and relates to a reagent box for detecting chromosome aneuploidy and a preparing method and an application thereof. The reagent box comprises a first reagent, a third reagent with purification magnetic beads or a second reagent, and a third reagent with the purification magnetic beads; the first reagent comprises a breaking repairing buffer solution, a breaking repairing enzyme, a dATP, a dNTP, a DNA polymerase I and a rtaq enzyme; the second reagent comprises the breaking repairing buffer solution, the breaking repairing enzyme, the dATP, the dNTP, a klenow enzyme and the rtaq enzyme; the third reagent comprises T4 DNA ligase, a PNK buffer solution and a junction. According to the reagent box, through an efficient and precise building database method, chromosome abnormity is detected, the comparison rate is increased, the GC deviation is reduced, the dup rate is lowered, meanwhile 10bp fluctuation before sequencing can be reduced, and the situation of AT separation can be avoided.