The present invention relates to the method of establishing symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. Germinated tomato seedling is first soaked with agrolacterium suspension and inoculated to sucrose-agar culture medium for culture to induce hairy root; the tomato hairy root is then transferred to M culture medium and AM fungi spore is inoculated for dark culture at 25 deg.c; and the germinated hypha invades into root system to establish the symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. The present invention induces tomato hairy root and establishes the symbiotic relationship between AM fungus and tomato hairy root on bacteria-free culture medium. The system of the present invention may be used in the amplification of AM fungus to obtain bacteria-free AM fungus material and in the relevant research of AM fungus under bacteria-free condition.