The invention discloses a molecular standard sample for bull's eye rot bacteria on apples and a preparation method thereof, and belongs to the field of the research on plant quarantine technologies. The molecular standard sample for the bull's eye rot bacteria on the apples is prepared from high-purity deoxyribonucleic acid (DNA) extracted from bacterial strains of the bull's eye rot bacteria on the apples. The standard sample has the following physical and chemical properties: (1) the standard sample is pure white powder in shape; (2) the content in each tube is 5+/-0.5mu g; (3) the purity of DNA is that OD260/280 is equal to 1.8 to 2; and (4) the standard sample is easily soluble in water or a TE (Tris-EDTA) buffer solution. The molecular standard sample for the bull's eye rot bacteria on the apples is obtained by the following steps of: culturing the bacterial strains, which are taken raw materials, of the bull's eye rot bacteria on the apples in a large quantity under specific conditions, extracting the high-purity DNA, split-charging, and freeze-drying to obtain a molecular standard of the molecular standard sample for the bull's eye rot bacteria on the apples. The standard sample has excellent physical and chemical properties and can be detected by using a specific primer by a polymerase chain reaction (PCR) method, and the sensitivity is 4 to 10 times. Through detectiontests, the DNA of the standard sample has high integrity, uniformity and stability; and the standard used as a positive reference substance can be used for import and export detection so as to improve the accuracy of detection results.