The invention provides a kit for separating genome DNA by using magnetic balls and application thereof. The kit comprises magnetic balls, a magnetic frame, a genome DNA extraction reagent (lysing solution, binding solution, rinsing solution A, rinsing solution B and eluent) and specifications; the main steps of extracting the gene DNA comprise cell lysis, nucleic acid absorption, impurity removing and nucleic acid elution. The application of the kit for extracting the genome DNA does not need to use large-toxicity organic solvents of phenol, chloroform and the like, has good safety, and simple, fast and time-saving operation, and simultaneously can extract a plurality of samples. The kit can extract the genome DNA from materials of animals, plants, bacteria, fungus, blood, virus, animal source feed stuff, samples in the forensic medicine and the like, the DNA has high yield and purity, and the obtained genome DNA can be used for experiments such as PCR amplification, gene cloning, construction of genomic library, sequence measurement, molecular hybridization, molecular marking and the like. The kit can be stored at the temperature of 4 DEG C, also can be placed at room temperature, and is convenient to transport.