The invention discloses an extraction and purification method of total plant endophyte genome DNA for colony analysis. The method comprises the following steps of: shearing and grinding the surface bacteria removed fresh plant tissues into paste, soaking the plant tissues into sterilized phosphate buffer solution, performing thermostatic shaking for 1 hour in a table concentrator to separate out microbial cells from the plant tissues, standing, transferring the suspension to a centrifuge tube, performing centrifugal collection on microbial bacteria, then adding lysing solution, shaking and uniformly mixing the solution to release DNA from the cells, repeatedly extracting protein by chloroform-isoamylol, performing centrifugal sedimentation with isopropanol and washing with 70 percent ice ethanol, performing purification by using a DNA specific centrifugal adsorption column to obtain total crude extracted genome DNA, and finally, performing polymerase chain reaction (PCR) amplificationby using special primers to obtain high-quality plant endophyte specific DNA fragments for subsequent analysis. The invention has the advantages that: the method is simple and effective, high in quality, low in pollution and the like, and provides high-quality guarantee for comprehensively and completely researching a plant endophyte colony structure.