The invention relates to a method for acquisition of an oligonucleotide probe. The method comprises the following steps: downloading genomic sequence of crop species from a public database, filtering and finding out tandem repeat sequences, screening preliminarily found repetitive sequences, comparing residual tandem repeat sequence and known probe sequence, removing already used probe sequence, comparing residual screened tandem repeat sequences, carrying out probe design and synthesizing and verifying the designed probe sequence so as to obtain effective oligonucleotide sequence. According to the invention, cost is low and efficiency is high. The probe can be used for non-degenerated fluorescence in situ hybridization (ND-FISH) analysis of crop chromosome, determination of distribution of tandem repeat sequences represented by the probe on chromosome, understanding of the structure characteristics of chromosome and establishment of specific landmark of chromosome. Thus, specific chromosome or chromosome region of crops is identified, and the developed new oligonucleotide probe has specificity of chromosome or chromosome region.