The invention discloses a method for preparing coccidiosis oocysts without exogenous bacteria DNA contamination, and belongs to the biotechnical field. With use of secondary distilled water without microbial DNA contamination, a required reagent is prepared; the oocysts are purified by a 1.2 M sucrose flotation precipitation method; the oocysts after purification are subjected to double treatment by a 15% sodium hypochlorite solution and a cell lysate, and residual bacteria and other impurities are fully cracked; then exogenous bacteria DNA attached on oocyst walls are removed through TE and secondary distilled water repeated rinsing. Researches indicate that the oocysts treated by the method have high purity, an extracted genomic DNA has no exogenous bacteria DNA contamination through PCR detection, and the whole process has the advantages of simple operation and low cost, and does not have any effect on the oocysts. The providing of the method has important significance on coccidiosis oocyst high-purity genomic DNA extraction, gene cloning, whole genome (re)sequencing and researches on epigenetic inheritance regulation.