The invention belongs to the technical field of biology and relates to a preparation method of a chromosomal DNA suitable for spore-bearing fungus karyotype analysis. The method is characterized by comprising the steps of collecting conidia; preparing spore agarose plugs; preparing protoplast agarose plugs; and preparing complete chromosome agarose plugs. The method is simple in operation, and the high-concentration complete chromosomal DNA can be obtained and can be directly applied to fungus karyotype analysis and genetic breeding research.