The invention discloses a biological pesticide capable of killing ova and a preparation method and application thereof. The biological pesticide contains emamectin benzoate, escin and griffonia seed oil. The biological pesticide capable of killing the ova is prepared by mixing emamectin benzoate, escin, griffonia seed oil, a polyalcohol nonionic surfactant and carboxymethylcellulose, and adding water during stirring to form homogeneous and slight turbid emulsion. The biological pesticide has the effect of expelling mosquitoes and obvious effects of killing pests in the living environment of people, such as cockroaches, termites and ants, wherein the killing rate is over 95 percent; besides adult pests, the biological pesticide can also kill the ova to prevent the reproduction of the pests so as to greatly improve the pest killing effect, and overcomes the limitation that the main active ingredient in the formula, namely emamectin benzoate cannot kill the ova; and simultaneously, the biological pesticide can spread toxin to pest populations because of mutual lick habits of the ants and cockroaches.