The invention relates to the technical field of edible fungi, and particularly discloses a lentinus edodes strain and applications thereof, and a pesticide derived from the strain and a preparation method thereof. The preparation method comprises the following steps: cleaning and drying lentinus edodes sticks, and performing crushing treatment to obtain lentinus edodes stick powder; and mixing thepowder with an extraction solvent, performing extraction treatment and filtration to obtain a lentinus edodes stick extract liquid, namely the pesticide. The preparation method of the pesticide provided by the invention has the advantages of simple process, convenient operation, environmental protection, safety and reliability. The obtained pesticide has wide insecticidal spectrum, safety and high efficiency, has the characteristics of low toxicity, easy degradation, low residue and environmental friendliness, and is beneficial to reducing the use of chemical pesticides and ensuring food safety.