The present disclosure discloses a method for preparing vanillin by fermentation with eugenol as a substrate, preparing vanillin by a mixed fermentation of Penicillium simplicissimum OMK-68 and Bacillus sp. OMK-69, its potency reached 35 g/L, the mass conversion rate of eugenol reaches 83.3%. Penicillium simplicissimum OMK-68 in the present disclosure can use eugenol as a substrate for fermentation to prepare coniferyl alcohol, and its fermentation effect and substrate utilization rate are far better than wild-type Penicillium simplicissimum. The Bacillus sp. OMK-69 in the present disclosure can use coniferyl alcohol as a substrate for fermentation to prepare vanillin, and its fermentation effect and substrate utilization rate are far better than wild-type Bacillus sp.