The invention belongs to the field of ant-killing medicaments, and particularly provides an environment-friendly ant-killing bait formulation and a preparation method thereof. The environment-friendlyant-killing bait formulation comprises an ant-killing medicament and a filling bait. The ant-killing medicament comprises dinotefuran and flonicamid. The filling bait comprises flour, corn flour, butter, peanut oil and diatomite. During preparation, the diatomite is mixed with the butter and the peanut oil, the corn flour and water are added to form paste, the flour and the water are added to obtain dough, and the dough is baked; the dinotefuran and the flonicamid are dissolved in acetone to obtain the ant-killing medicament, and the filling bait is soaked in the ant-killing medicament, driedin an oven, and screened by a 10-18 mesh sieve after smashing. The environment-friendly ant-killing bait formulation is made from bioactivators with specific effect on growth and development of solenopsis invicta, is goal-oriented, does not harm non-target organisms easily, is harmless to the environment and efficient, and kills ants thoroughly.