The present disclosure provides a preparation method of eucalyptol emulsion and application thereof in biopesticides, belonging to the field of emulsion preparation. The method for preparing the eucalyptol emulsion includes the steps of: (1) adding eucalyptol into a cyclodextrin solution to obtain a mixed solution; (2) evenly mixing an emulsifier and a co-emulsifier to obtain a mixed solution; (3) adding the solution obtained in step (2) and water into the solution obtained in step (1), emulsifying to obtain the eucalyptol emulsion. In steps (1), (2) and (3), the mass percents of substances from which the eucalyptol emulsion is prepared are as follows: 0.5%-1% of cyclodextrin, 2.5%-25% of the eucalyptol, 1.25%-15% of the emulsifier, 0.3%-3.75% of the co-emulsifier and 55.3%-95.5% of the water, and the sum thereof is 100%. The emulsion is extremely stable without stratification after being placed at 20° C., 40° C. and 60° C. for 30 days and also has a good insecticidal effect.