The invention discloses application of heterorhabditis bacteriophora to biological control of underground insects of greenhouse vegetables. The heterorhabditis bacteriophora is obtained through separation, and fresh chicken internal organs are adopted or chicken gizzards and gallbladders are removed; 20%w/v of water and 10%-20% of vegetable oil are added, and the mixture is homogenized and sterilized to be used as a culture medium for culturing and breeding entomopathogenic nematodes; nematode suspension liquid with the concentration of 10000Ijs per seedling is prepared and is filled to roots of the seedlings; after application, the fatality rate of mosquito and fly larvae is 100%, and the fatality rate of large-size underground insects including grubs, mole crickets and the like is 50% or more; the heterorhabditis bacteriophora has the acting effects of no toxin and no pollution, and capability of keeping a long effect after one time of application; the heterorhabditis bacteriophora has active searching and expanding propagation capabilities, so that the heterorhabditis bacteriophora has the acting effect on underground insect larvae which are difficult to prevent and control and the acting effect is better than that of other medicines; and the heterorhabditis bacteriophora has a wide application prospect on production of green and healthy greenhouse vegetables.