The invention discloses an environment-friendly biological insecticide formula and a preparation method thereof. The environment-friendly biological insecticide formula consists of tobacco leaves, maidenhair, bunge pricklyash leaves, tropic ageratum herbs, folia perillae acutae, cephalotaxus fortunei, cortex meliae, asparagus officinalis, rotenone, fructus toosendan, alcohol, ailanthus altissima leaves, carolina jessamine, divaricate strophanthus seeds, geranium wilfordii, castor bean shells, derris trifoliata, melia azedarach leaves, pyrethrum, sweet wormwood, garlics, peppers, ginger, root barks of tree peony and sweet wormwood. Compared with the prior art, the environment-friendly biological insecticide formula is prepared by adopting pure bio-fermentation, is a non-chemical synthetic insecticide, plays a role in protecting the environment and cannot cause harm to a human body after being absorbed by plants, thereby having popularization and application values.