Abstract: Rhopalosiphum nymphaeae is a major insect pest of aquatic vegetables and is capable of causing yield losses exceeding 50% under severe infestation. In recent years, there has been an increasing number of reports on odorant-binding proteins (OBPs) mediated pesticide resistance in insects. To elucidate the binding characteristics of R. nymphaeae OBPs towards pesticides, this study cloned RnymOBP9 gene. An integrated strategy involving phylogenetic analysis, quantitative real-time PCR (RT-qPCR), and fluorescent competitive binding assays was employed to investigate its tissue-specific expression profiles and binding affinity with eight pesticides and the aphid alarm pheromone, (E)-β-farnesene (EBF). Results showed that RnymOBP9 gene (GenBank accession number: PX123855) contains an open reading frame (ORF) of 384 bp, encoding a protein of 127 amino acids. Phylogenetic analysis revealed that RnymOBP9 is closely related to other aphid OBP9 orthologs. Tissue expression profiling indicated that RnymOBP9 is expressed in multiple tissues, with notably high and specific expression in the antennae. When the recombinant RnymOBP9 protein was bound to the N-phenyl-1-naphthylamine (1-NPN) fluorescent probe, the addition of imidacloprid, dinotefuran, and EBF reduced the relative fluorescence intensity (RFI) to below 50%. Notably, EBF exhibited the strongest binding affinities to OBP9, with dissociation constants (K_d) of (6.389 ± 0.596) μmol/L, followed by dinotefuran, with a K_d of (10.51 ± 0.754) μmol/L. These findings demonstrate that RnymOBP9 binds both EBF and synthetic insecticides, suggesting its potential involvement in key biological processes such as chemical signal recognition and the mediation of insecticide resistance in R. nymphaeae.