A preparation method for in-situ hybridization probes as follows: fragmenting objective DNAs, recovering 150-600 bp fragments, and after an enzyme modification, ligating, at intervals, the fragments with DNA adaptors containing restriction enzyme site sequences to large DNA loops and long chains; obtaining and labeling a large amount of DNAs in step A or B: A. isothermal amplifying, adding a single nucleotide substrate with a marker when amplifying, to obtain a DNA product with a marker; or B. isothermal amplifying, doping a single nucleotide substrate with a marker to the obtained product with a nick translation or random primer method, to obtain a DNA product with a marker; and digesting the DNA product with the marker by using corresponding restriction enzyme, to obtain in-situ hybridization probes with lengths of 150-600 bp. The method of the present invention accurately controls length range of the probes, reduces production cost, and improves product quality.