The invention relates to a natural biological pesticide fertilizer, a preparation method and a use method thereof. Through detection on the basis of a chromatography and mass spectrometry combined measuring process, the natural biological pesticide fertilizer provided by the invention comprises the following components: 8-52% of acetic acid, 1-4.3% of formic acid, 0.6-3.2% of propionic acid, 0.02-0.1% of hydroxyacetone, 0.4-2% of isoquinoline, 0.2-0.9% of guaiacol, 0.8-2.3% of p-cresol, 0.3-1.8% of m-cresol and the balance of water. The invention has the beneficial effects that the natural biological pesticide fertilizer is easy to dissolve in water, can be easily absorbed by plants, is beneficial to sterilization, parasite expelling and inhibition of plant diseases and insect pests, and is capable of enhancing immunity system of plants, strengthening roots, boosting seedlings, promoting plant absorption, regulating plant metabolism, improving soil and promoting quality and stress resistance of crops; the natural biological pesticide fertilizer can be widely applied to economic crops, such as cereals, vegetables, melon and fruit, tea leaves, edible mushrooms, garden plants and Chinese herbal medicines; the natural biological pesticide fertilizer can be used for treating plant seeds, dipping roots, spraying, performing drop irrigation, watering, and preventing and controlling pests.