The invention discloses a flaking method of petunia chromosome. The method comprises the following steps: getting a petunia young bud at 8:00-10:00 in the morning of a clear day, placing in 0.002mol.L<-1> 8-hydroxyquinoline aqueous solution for pre-treating at low temperature, transferring into a Carnoy's fluid to be fixed, performing low-permeation through 0.075mol.L<-1> potassium chloride, and then water-bathing at 60 DEG.C in 1mol.L<-1> HCl solution for decomposing, washing and then low-permeating through the distilled water, and staining through aceto-carmine staining solution, transferring the young bud onto a glass slide, cutting to get a pistil stigma part, flaking and performing microscopy. The bud size, the pretreatment method, the decomposition and staining method and time and the flaking processes of petunias with different ploidy at optimal sampling time are determined, the obtained chromosome is uniform in dispersion, easy to count and easy for performing karyotype analysis, the operation is simple and convenient, the efficiency is high, and the method has a good application prospect.