The invention belongs to the field of cytobiology and molecular biology studies and particularly relates to oligonucleotide dye liquor and application and a usage method thereof. The oligonucleotide dye liquor comprises YMPNS-Centc69-1, mknob-2, YMPNS-MR68-3 and (ACT)<10> which are four modified oligonucleotide probes, mixing is conducted according to a certain ratio, dilution is conducted through 2xSSC, and thus the oligonucleotide dye liquor is formed. 8 [mu]l of the oligonucleotide dye liquor is added onto each non-denatured chromosome piece, 2.5 [mu]l of 50%w/v dextran sulfate is added, hybridization is conducted in an incubator at the temperature of 37 DEG C for two hours, then washing is conducted in 2xSSC at the temperature of 42 DEG C for five minutes, and observation can be conducted below a fluorescence microscope after over dyeing through DAPI. The oligonucleotide dye liquor can be developed into a commodity to be used for fast chromosome identification of corn and affinity species of the corn.