: The whitefly, Bemisia tabaci, is an important invasive economic pest of agricultural crops worldwide. β-ionone has a significant oviposition repellent effect against B. tabaci, but the olfactory molecular mechanism of this insect for recognizing β-ionone is unclear. To clarify the binding properties of odorant-binding proteins (OBPs) with β-ionone, we performed gene cloning, evolution analysis, bacterial expression, fluorescence competitive binding assay, and molecular docking to study the binding function of OBP1 and OBP4 on β-ionone. The results showed that after the OBP1 and OBP4 proteins were recombined, the compound β-ionone exhibited a reduction in the fluorescence binding affinity to <50%, with a dissociation constant of 5.15 and 3.62 μM for OBP1 and OBP4, respectively. Our data indicate that β-ionone has high affinity for OBP1 and OBP4, which play a crucial role in the identification of oviposition sites in B. tabaci. The findings of this study suggest that whiteflies employ β-ionone compound in the selection of the suitable egg-laying sites on host plants during the oviposition behavior.