A preparation method for in-situ hybridization probes, characterized by including the following steps: fragmenting and selective recovering of objective DNAs: fragmenting the objective DNAs, and selectively recovering 150-600bp small fragments; enzyme modification processing: performing an enzyme modification processing on two ends of 150-600bp small fragments, to make the two ends blunt, then 5' end phosphate added and 3' end dA added; ligating into large loops and long chains: ligating the above processed small fragments and DNA adaptors at intervals to large DNA loops and long chains, wherein the adaptor has restriction enzyme site sequences, phosphate at the 5' end and dT at the 3' end; obtaining and labeling of a large amount of DNAs: A. isothermal amplifying, doping a single nucleotide substrate with marker when amplifying, to obtain a DNA product with the marker; or B. isothermal amplifying, doping a nucleotide substrate with a marker to the obtained product with a nick translation or random primer method, to obtain a DNA product with the marker; enzyme digesting: digesting the DNA product with the marker by adopting a DNA restriction enzyme corresponding to a base sequence of the above DNA adaptor, wherein a length range of the enzyme digested product is 150-600bp; and obtaining a probe: if the marker in the DNA product with the marker is a direct marker, the obtained enzyme digested product being the in-situ hybridization probe; and if the marker in the DNA product with the marker is an indirect marker, the obtained enzyme digested product being coupled with the direct marker having corresponding reactivity, thereby obtaining the in-situ hybridization probe. fragmenting and selective recovering of objective DNAs: fragmenting the objective DNAs, and selectively recovering 150-600bp small fragments; enzyme modification processing: performing an enzyme modification processing on two ends of 150-600bp small fragments, to make the two ends blunt, then 5' end phosphate added and 3' end dA added; ligating into large loops and long chains: ligating the above processed small fragments and DNA adaptors at intervals to large DNA loops and long chains, wherein the adaptor has restriction enzyme site sequences, phosphate at the 5' end and dT at the 3' end; obtaining and labeling of a large amount of DNAs: A. isothermal amplifying, doping a single nucleotide substrate with marker when amplifying, to obtain a DNA product with the marker; A. isothermal amplifying, doping a single nucleotide substrate with marker when amplifying, to obtain a DNA product with the marker; or B. isothermal amplifying, doping a nucleotide substrate with a marker to the obtained product with a nick translation or random primer method, to obtain a DNA product with the marker; enzyme digesting: digesting the DNA product with the marker by adopting a DNA restriction enzyme corresponding to a base sequence of the above DNA adaptor, wherein a length range of the enzyme digested product is 150-600bp; and obtaining a probe: if the marker in the DNA product with the marker is a direct marker, the obtained enzyme digested product being the in-situ hybridization probe; and if the marker in the DNA product with the marker is an indirect marker, the obtained enzyme digested product being coupled with the direct marker having corresponding reactivity, thereby obtaining the in-situ hybridization probe. if the marker in the DNA product with the marker is a direct marker, the obtained enzyme digested product being the in-situ hybridization probe; and if the marker in the DNA product with the marker is an indirect marker, the obtained enzyme digested product being coupled with the direct marker having corresponding reactivity, thereby obtaining the in-situ hybridization probe.